Method for determining the risk of developing normal tension glaucoma in Koreans specific by apolipoprotein E gene polymorphism analysis

Abstract

The present invention relates to a method for determining the risk of developing normal tension glaucoma specific to Koreans by analyzing apolipoprotein E gene polymorphisms. Specifically, the invention relates to a method for identifying single nucleotide polymorphisms (SNPs) from a biological sample, a biomarker composition for identifying normal tension glaucoma in Koreans containing 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 the analysis of apolipoprotein E gene 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 determining the risk of developing normal-tension glaucoma specific to Koreans by analyzing apolipoprotein E gene 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] Meanwhile, the impact of single nucleotide polymorphisms (SNPs) of apolipoprotein E (APO E) on open-angle glaucoma and normal-tension glaucoma is still controversial, and according to existing reports, the APOE e3 / e4 ​​alleles are already known to have a significant effect on Alzheimer's disease.

[0005] Accordingly, the inventors investigated the association between the incidence of normal-tension glaucoma and the APOE haplotype in a Korean cohort and confirmed that there is a significant association, thereby completing 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 rs449647, rs769446, rs405509, rs429358, and rs7412 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 rs449647, rs769446, rs405509, rs429358, and rs7412.

[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 present invention provides a method for identifying one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of rs449647, rs769446, rs405509, rs429358, and rs7412 from a biological sample in order to provide information necessary for predicting the risk of developing normal tension glaucoma in Koreans.

[0013] 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.

[0014] 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.

[0015] 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 rs2946370. 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.

[0016] The rs449647, rs769446, rs405509, rs429358, and rs7412 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 / ).

[0017] According to one embodiment of the present invention, if rs769446C>T is located on chromosome 19 of apolipoprotein E (APO E), it can be determined that the risk of normal tension glaucoma (NTG) is increased.

[0018] In the present invention, the single nucleotide polymorphism (SNP) may be located on chromosome 19 of the apolipoprotein E (APO E) gene.

[0019] In the present invention, the biological sample may be blood, tissue, cell, serum, plasma, saliva, urine, etc., but is not limited thereto.

[0020] In the present invention, the subject of the biological sample may be a Korean.

[0021] In the present invention, the single nucleotide polymorphism (SNP) may be determined using a haplotype.

[0022] 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 rs449647, rs769446, rs405509, rs429358, and rs7412.

[0023] 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.

[0024] 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.

[0025] In the present invention, the primer or probe that specifically binds to the polynucleotide or its complementary polynucleotide is allele-specific.

[0026] 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.

[0027] 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.

[0028] 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).

[0029] 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.

[0030] 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.

[0031] 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.

[0032] In addition, the present invention provides a kit for predicting the risk of developing normal tension glaucoma, comprising the above composition.

[0033] 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.

[0034] 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

[0035] 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 haplotypes of single nucleotide polymorphisms. Brief explanation of the drawing

[0036] Figure 1 shows five SNPs displayed in the UCSC Genome Browser. Specific details for implementing the invention

[0037] 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.

[0039] Example 1. Experimental Method

[0041] 1.1 Selection of Research Group

[0043] Unrelated NTG patients and healthy controls were recruited from the Department of Ophthalmology at Uijeongbu St. Mary's Hospital in South Korea. All subjects received written consent after being informed of the purpose of the 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 (Approval No.: UC16SISE0060) and conducted in accordance with the principles of the Declaration of Helsinki. For the haplotype analysis, 178 NTG patients recruited from Uijeongbu St. Mary's Hospital and 32,858 glaucoma-free controls from the KoGES cohort were utilized.

[0044] The group of normal-tension glaucoma patients recruited by the Department of Ophthalmology at Uijeongbu St. Mary's Hospital was selected through the following process. 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.

[0045] 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%.

[0046] 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.

[0048] 1.2 Sample Preparation and Allele Identification

[0050] Genomic DNA was isolated from blood using the MG Blood Genomic DNA Extraction SV kit (MGmed, Seoul, Korea). Five SNPs each at the APOE locus (four types including rs449647, see Table 1) were analyzed in this invention.

[0051] Genotyping analysis of 19 polymorphisms including rs2946370 was performed using 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, with the QuantStudio real-time polymerase chain reaction system (Applied Biosystems).

[0052] For the control group extracted from KoGES, information on 5 APOE SNPs was extracted from all control groups in the genotypes that had completed the Axiom Biobank Array version 1.1 (Korean-chip Microarray sequencing) method and NARD2 imputation, and used for haplotype analysis.

[0054] 1.3 Extraction of Independent Single Nucleotide Polymorphism (SNP) Factors

[0056] Based on a genome-wide association study (GWAS) of normal-tension glaucoma, five independent APOE SNP genotypes of genome-level significance were extracted from both the patient and control groups. Table 1 shows the five SNPs used in the construction of the PRS of the present invention.

[0058] No. rsID (hg38) CHR POS REF ALT GENE(hg38) 1 rs449647 19 44905307 A T APOE promoterChr19:44,905,791 - 44,909,393(sequence number 1) 2 rs769446 19 44905371 T C APOE promoterChr19:44,905,791 - 44,909,393 3 rs405509 19 44905579 T G APOE promoterChr19:44,905,791 - 44,909,393 4 rs429358 19 44908684 T C APOE exonChr19:44,905,791 - 44,909,393(sequence number 2) 5 rs7412 19 44908822 C T APOE exonChr19:44,905,791 - 44,909,393

[0060] Example 2. Experimental Results

[0062] Haplotype analysis was performed on five SNPs located on chromosome 19 of apolipoprotein E (APO E) (three promoter SNPs rs449647, rs769446, rs405509 and two exonic SNPs rs429358, rs7412) using SHE-sis web-based software (Analysis tool for random samples, by YongYong Shi. (bio-x.cn)) (Fig. 1).

[0064] For the control group, the average age was 53.8 ± 8.0 years, and the male-female ratio was 66% female, which was confirmed in the KoGES database. Along with p-values, the number of 5 SNP alleles, allele frequencies, and genotype frequencies showed that the rs769446 genotype differed significantly between the cases and the control group. Among the 5 SNPs mentioned above, the second promoter SNP rs769446 minor allele C was a protective allele for normal-tension glaucoma (Fig. 2). The frequencies of the 5 APOE SNP alleles and genotypes in the cases and control group are shown in Table 2.

[0066] Allele Frequency, % Polymorphism NTG, n=178 * Control, n=32858 * p-value (NTG vs.Control) rs449647 Allele 0.91 A 345 (96.9) 63752 (97.0) T 11 (3.1) 1964 (3.0) Genotype 0.09 AA 168 (94.6) 30922 (94.1) AT 9 (4.9) 1908 (5.8) TT 1 (0.5) 28 (0.1) rs769446 Allele 0.008 T 344 (96.6) 61128 (93.0) C 12 (3.4) 4588 (7.0) Genotype 0.009 TT 166 (93.3) 28429 (86.5) TC 12 (6.7) 4270 (13.0) CC 0 (0) 159 (0.5) rs405509 Allele 0.11 T 268 (75.3) 46931 (71.4) G 88 (24.7) 18785 (28.6) Genotype 0.25 TT 102 (57.3) 16801 (51.1) TG 64 (36.0) 13329 (40.6) GG 12 (6.7) 2728 (8.3) rs429358 Allele 0.96 T 323 (90.7) 59679 (90.8) C 33 (9.3) 6037 (9.2) Genotype 0.95 TT 147 (82.6) 27117 (82.5) TC 29 (16.3) 5445 (16.6) CC 2 (1.1) 296 (0.9) rs7412 Allele 0.08 C 341 (95.8) 61421 (93.5) T 15 (4.2) 4295 (6.5) Genotype 0.09 CC 163 (91.6) 28704 (87.4) CT 15 (8.4) 4013 (12.2) TT 0 (0) 141 (0.4)

[0068] In addition, haplotype analysis confirmed that haplotypes ACGTT (protective) and ATGTT (risk) had statistically significant p-values ​​(Fig. 3). The five haplotypes are listed in the order mentioned above. The results of the haplotype analysis using SHE-sis software are shown in Table 3.

[0070] Haplotype analysis (SHEsis software) NTG vs Control Haplotype Case(freq) Control(freq) Pearson's p Odds ratio 95%CI N=178 N=32858 ACGTT 0.031 0.063 0.011205 0.467 [0.256~0.853] ATGTC 0.19 0.206 0.438866 0.901 [0.691~1.174] ATGTT 0.011 0.002 3.93E-05 6.252 [2.299~17.003] ATTCC 0.073 0.072 0.961666 1.01 [0.677~1.505] ATTTC 0.66 0.619 0.147672 1.177 [0.944~1.467] TTGTC 0.012 0.011 0.839924 1.105 [0.420~2.902] TTTCC 0.019 0.018 0.872858 1.064 [0.497~2.277] ------------- The 5 single nucleotide polymorphisms used for haplotype analysis are ordered in the following - rs449647, rs769446, rs405509, rs429358, rs7412 frequency<0.01 in both control & case has been dropped.

[0072] These results provide a new perspective on the role of APOE SNPs in normal-tension glaucoma and demonstrate that 5 SNP haplotype analysis can be used in future clinical settings for people with specific haplotypes, confirming that the protective effect of these specific haplotypes can be usefully employed to determine the clinical outcomes and risks of glaucoma.

[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 all single nucleotide polymorphisms (SNPs) of rs449647, rs769446, rs405509, rs429358, and rs7412 from a biological sample. Claim 2 A method according to claim 1, characterized in that the single nucleotide polymorphism (SNP) is located in the apolipoprotein E (APO E) gene of chromosome 19. Claim 3 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 4 A method according to claim 1, characterized in that the subject of the biological sample is a Korean. Claim 5 A method according to claim 1, characterized in that the single nucleotide polymorphism (SNP) is determined using a haplotype. Claim 6 A biomarker composition for identifying normal tension glaucoma in Koreans containing all single nucleotide polymorphisms (SNPs) of rs449647, rs769446, rs405509, rs429358, and rs7412. Claim 7 A composition for predicting the risk of developing normal tension glaucoma, comprising a preparation capable of detecting the biomarker composition of claim 6. Claim 8 A kit for predicting the risk of developing normal tension glaucoma, comprising the composition of claim 7.

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