KR20240172634A - Method for determining the risk of developing normal tension glaucoma in Koreans specific by polymorphism - Google Patents

Abstract

The present invention relates to a method for analyzing the risk of developing normal tension glaucoma in Koreans using a single nucleotide polymorphism, and more particularly, to a method for determining at least one single nucleotide polymorphism (SNP) selected from the group consisting of rs10483727 and rs33912345 from a biological sample, a biomarker composition for determining normal tension glaucoma in Koreans including the single nucleotide polymorphism (SNP), a composition for predicting the risk of developing normal tension glaucoma, comprising an agent 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 for predicting the risk of developing normal tension glaucoma in Koreans based on a single nucleotide polymorphism can be provided. In particular, when the kit of the present invention is used, such information can be provided more easily.

Description

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

The present invention relates to a method for analyzing the risk of developing normal tension glaucoma in Koreans using single nucleotide polymorphisms.

Glaucoma is a degenerative and progressive optic neuropathy characterized by glaucomatous optic nerve damage with visual field defects. Glaucoma is one of the leading causes of visual field loss and blindness worldwide. Recent studies have shown that open-angle glaucoma (OAG) is phenotypically and genetically heterogeneous and is polygenic in nature.

Increased intraocular pressure (IOP) is not an essential factor in the diagnosis of glaucoma. In Koreans, normal tension glaucoma (NTG) is the most common subtype of glaucoma. Studies of various ethnic groups have shown that NTG and NTG share some genetic risk factors, but the association with glaucoma may differ depending on genetic variation.

Previous studies have reported that the SIX1/SIX6 locus is associated with open-angle glaucoma (OAG) in various ethnicities. However, the definition of OAG patients varied among studies, and the association of the SIX1/SIX6 locus with normal tension glaucoma (NTG) in Koreans remains uncertain.

Accordingly, the inventors of the present invention investigated the association between the incidence of normal-tension glaucoma and the SIX1/SIX6 gene locus in a Korean cohort and attempted to use the association to predict or diagnose normal-tension glaucoma. They confirmed that SIX1/SIX6, an OAG-related SNP, was significantly associated with normal-tension glaucoma, and completed the present invention.

Republic of Korea Publication Patent No. 10-2023-0075213 (2023.05.31)

The purpose of the present invention is to provide a method for determining at least one single nucleotide polymorphism (SNP) 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.

Another object of the present invention is to provide a biomarker composition for determining normal tension glaucoma in Koreans, comprising at least one single nucleotide polymorphism (SNP) selected from the group consisting of rs10483727 and rs33912345.

Another object 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.

Another object of the present invention is to provide a kit for predicting the risk of developing normal tension glaucoma, comprising the composition.

Other objects and advantages of the present invention will become more apparent from the detailed description of the invention, the claims and the drawings which follow.

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

In addition, the rs33912345 polymorphism was significantly associated 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). 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 NTG cases, controls, and alleles under dominant or recessive models (all P >0.05). Therefore, we confirmed that the SIX1-SIX6 loci rs10483727 and rs33912345 polymorphisms were significantly associated with the risk of NTG in Koreans.

The present invention provides a method for determining at least one single nucleotide polymorphism (SNP) 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.

In the present invention, polymorphism refers to a case where two or more alleles exist at one locus, and among the polymorphic sites, a single nucleotide polymorphism (SNP) that differs only by a single base depending on the individual is called a single nucleotide polymorphism. A preferable 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 for a polymorphic marker means that there is an association for one or more specific alleles of a specific polymorphic marker. A marker may include any variant form of an allele found in a genome, including a single nucleotide polymorphism (SNP), a microsatellite, an insertion, a deletion, a duplication, and a translocation.

In the present invention, an allele or biallelic trait refers to multiple types of a gene existing at the same locus of a homologous chromosome. An allele is also used to indicate polymorphism, for example, a SNP has two types of alleles.

In the present invention, rs_id means 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 described in such a table means a SNP marker, which is a polymorphic marker of the present invention. Those skilled in the art will be able to easily confirm the location and sequence of the SNP using the rs_id.

In the present invention, rs10483727 and rs33912345, which are used 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://http://www.ncbi.nlm.nih.gov/snp/).

Meanwhile, carriers of the AA genotype for rs33912345 or the CC genotype for rs10483727 were determined to have a reduced risk of normal tension glaucoma (NTG). Specifically, minor alleles for two SNPs in SIX1/SIX6 showed protective effects, and were confirmed 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 the rs33912345 appear at a higher rate in the control group than in the patient group, it is determined that the Minor allele (A) has a protective effect, and since CC genotype carriers for the rs10483727 appear at a higher rate in the control group than in the patient group, it is determined that the Minor allele (C) has a protective effect.

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

In the present invention, the single nucleotide polymorphism (SNP) may be located in the intergenic region between the SIX1 and SIX6 gene loci.

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

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

In addition, the present invention provides a biomarker composition for determining normal tension glaucoma in Koreans, comprising at least one single nucleotide polymorphism (SNP) selected from the group consisting of rs10483727 and rs33912345.

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

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

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

In the present invention, a primer refers to a short nucleic acid sequence having a short free hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. The primer of the present invention can be chemically synthesized using a method known in the art, such as, for example, a phosphoramidite solid support method.

In the present invention, the probe refers to a nucleic acid fragment such as RNA or DNA consisting of several to several hundred 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.

In the present invention, the probe may be labeled with a detectable substance, for example, a radioactive label that provides a suitable signal and has a sufficient half-life. The labeled probe may be hybridized to a nucleic acid on a solid support as described in the literature (Sambook et al., Molecular Cloning, A Laboratory Manual, 1989).

In the present invention, the probe is an allele-specific probe, and since there is a polymorphic site in the nucleic acid fragment, it may hybridize to a nucleic acid fragment including one allele but not to a nucleic acid fragment including the other allele. In this case, the hybridization conditions must be sufficiently stringent so that there is a significant difference in hybridization intensity between the alleles and hybridize to only one of the alleles. Preferably, the probe may be single-stranded for maximum efficiency in hybridization, but is not limited thereto.

In the present invention, 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 is amplified using a method known in the art. In addition, detection of a specific nucleic acid using a probe can be performed by contacting a sample nucleic acid with a probe under suitable conditions and then confirming the presence of a hybridized nucleic acid.

In the present invention, methods for detecting a specific nucleic acid using the probe or primer include, but are not limited to, polymerase chain reaction (PCR), DNA sequencing, RT-PCR, primer extension (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 (Rust et al., Nucl Acids Res, 6, 3623-3629, 1993), RNase mismatch cleavage (Myers et al., Science, 230, 1242-1246, 1985), single strand conformation lymorphism (Ori-ta et al., Pro Nat Acad Sci USA, 87, 8923-8927, 1990). Sci USA, 86, 2766-2770, 1989), simultaneous SSCP and heteroduplex analysis (Lee et al., Mol Cells, 5:668-672, 1995), denaturing gradient gel electrophoresis (DGGE, Cariello et al., Am J Hum Genet, 42, 726-734, 1988), denaturing high pressure liquid chromatography (Underhill et al., Genome Res, 7, 996-1005, 1997), hybridization reactions, DNA chips, etc. Examples of such 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.

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

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

In the present invention, the kit may be a kit including essential elements necessary for performing PCR. In addition to a specific polynucleotide, primer or probe for the polymorphic marker, the PCR kit may include a test tube or other appropriate container, a reaction buffer (with various pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNAse inhibitor, DEPC water, and sterile water.

According to the present invention, there is an advantage in that it can be usefully used to predict the risk of developing normal tension glaucoma in Koreans based on single nucleotide polymorphisms.

Hereinafter, in order to help understand the present invention, examples will be given and described in detail. However, the following examples are only intended to illustrate 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 a person having average knowledge in the art.

Example 1. Experimental method

1.1 Study group selection

Unrelated NTG patients and healthy controls were recruited from the Department of Ophthalmology, Uijeongbu St. Mary's Hospital, Republic of Korea. All subjects gave written informed consent after being informed of the purpose of genetic testing 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, Republic of Korea (Approval Number: UC16SISE0060), and was conducted in accordance with the principles of the Declaration of Helsinki.

A thorough ophthalmologic examination, including optic nerve head examination, was performed on all participants. Inclusion criteria were as follows: having a characteristic glaucomatous optic nerve head as defined by VCDR ≥0.6; diffuse or focal neuroretinal rim thinning (e.g., notching or acquired optic disc lesions) and/or retinal nerve fiber layer (RNFL) defects; glaucomatous visual field defects as measured by a Humphrey automated field analyzer (Zeiss Humphrey Systems, Dublin, CA); open angle structure; and medication-free IO1 ≤21 mm Hg at the initial examination and repeat measurements on different days.

Glaucomatous visual field defect was defined as a cluster of three or more points with a probability <5% or a probability <5% on the pattern deviation map in at least one hemifield with at least one point with a probability <1%, and a field outcome of “normal limits on the external glaucoma hemifield test” or a pattern standard deviation with a probability <5%.

A total of 210 NTG patients (80 males and 130 females) with a mean age of 67.15±12.8 years met the inclusion criteria. Patients with closed-angle glaucoma, pigmentary glaucoma, uveitic glaucoma, neovascular glaucoma, or a history of trauma were excluded. Control subjects (117 patients, 46 males and 71 females, mean age 70.3±10.9 years) met the following criteria at recruitment: normal optic nerve head; normal IOP (≤21 mmHg); no history of elevated IOP; and no family history of glaucoma.

1.2 Sample preparation and allele determination

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) of the SIX1/SIX6 locus were analyzed in the present invention.

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

1.3 Statistical Analysis

Statistical analyses were 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. Frequencies and odds ratios (ORs) with 95% confidence intervals (CIs) for all polymorphic SNPs were calculated. Comparisons of genotype and allele frequencies were performed using the χ2 test or Fisher exact test. Allelic associations were assessed by logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (CIs).

SNP associations were tested using three genetic models: a dominant model [common allele homozygote (AA) vs. heterozygote (AB) + minor allele homozygote (BB)], a recessive model (AA+AB vs. BB), and an allelic 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.

Example 2. Experimental Results

To evaluate the possible association between NTG and SIX1/SIX6 loci, we performed genotyping of four SNPs in a Korean cohort. The study included 327 subjects (210 NTG patients and 117 healthy controls). For each SNP, all genotypes were in Hardy-Weinberg equilibrium in the control group (Table 1). Table 1 shows general information about the four SNPs.

Additionally, the association results of the four SNPs in the allelic model, dominant model, and recessive model are summarized in Table 2.

The 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 the significant association between rs33912345 and NTG was not found when the dominant or allelic model was used (P=0.204 or 0.064, respectively, Table 2).

The significant association between rs10483727 and NTG was confirmed using the 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 the significant association between rs10483727 and NTG was not detected using the dominant model (P=0.236, Table 2).

Genetic linkage analysis for rs12436579 and rs2179970 did not reveal statistically significant differences in genotype distributions between NTG patients and control subjects in allelic, dominant, or recessive models (all P > 0.05 Table 2 ).

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

While the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A method for determining one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of rs10483727 and rs33912345 from a biological sample to provide information necessary for predicting the risk of developing normal tension glaucoma in Koreans. In the first paragraph,
A method wherein the single nucleotide polymorphism of rs33912345 is determined by a recessive model, and the single nucleotide polymorphism of rs10483727 is determined by an allelic model or a dominant model. In the first paragraph,
A method wherein the risk of normal tension glaucoma is determined to be reduced in AA genotype carriers for rs33912345 or CC genotype carriers for rs10483727. In the first paragraph,
A method wherein the above single nucleotide polymorphism (SNP) is located in the intergenic region between the SIX1 and SIX6 loci. In the first paragraph,
A method, characterized in that the biological sample is any one selected from the group consisting of blood, tissue, cells, serum, plasma, saliva, and urine. In the first paragraph,
A method, characterized in that the subject of the above biological sample is Korean. A biomarker composition for determining normal tension glaucoma in Koreans, comprising at least one single nucleotide polymorphism (SNP) selected from the group consisting of rs10483727 and rs33912345. A composition for predicting the risk of developing normal tension glaucoma, comprising a preparation capable of detecting the biomarker composition of claim 7. A kit for predicting the risk of developing normal tension glaucoma, comprising the composition of claim 8.