WO2025058147A1 - Method for determining risk of developing high-risk open-angle glaucoma specific to koreans by polygenic risk score analysis - Google Patents

Abstract

The present invention relates to a method for determining the risk of developing high-risk open-angle glaucoma specific to Koreans by polygenic risk score analysis and, in particular, to: a method for determining single nucleotide polymorphism (SNP) from a biological sample; a biomarker composition for determining open-angle glaucoma in Koreans, comprising SNP; a composition for predicting the risk of developing open-angle glaucoma, comprising an agent for detecting the biomarker composition; and a kit for predicting the risk of developing open-angle glaucoma, comprising the composition. According to the present invention, it is possible to provide information useful in predicting the risk of developing open-angle glaucoma in Koreans on the basis of polygenic risk scores. In particular, such information can be more readily provided by using the kit of the present invention.

Description

Korean-specific high-risk open-angle glaucoma risk assessment method using polygenic risk score analysis

The present invention relates to a method for determining the risk of developing high-risk open-angle glaucoma in Koreans using multi-gene risk score analysis.

Glaucoma is not caused by daily lifestyle, is not contagious, and is not life-threatening, but it is the most common eye disease that causes blindness along with diabetic retinopathy. In particular, once glaucoma occurs, the optic nerve that has already been damaged cannot be restored, so early diagnosis and early treatment are important. Currently, many ophthalmologists are researching to identify the cause and provide effective treatment, but the exact cause of glaucoma is still unknown.

Specifically, 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.

Although there have been some previous studies on the relationship between glaucoma and genetic loci, the specific relationship is still uncertain, and the prediction method using polygenic risk score analysis based on the genome-wide association study (GWAS) study on open-angle glaucoma is insufficient in East Asians, who have a high incidence of glaucoma.

Accordingly, the inventors of the present invention investigated the association between the incidence of high-risk glaucoma and genetic loci in a Korean cohort and confirmed a significant association, thereby completing the present invention.

The purpose of the present invention is to provide information necessary for predicting the risk of developing open angle glaucoma in Koreans, and to provide at least one selected from the group consisting of rs2946370, rs66500121, rs4308305, rs9853115, rs28795989, rs61275591, rs57111852, rs2745572, rs2526101, rs1412829, rs2472494, rs3829849, rs61870251, rs11024102, rs4303192, rs58073046, rs35155027, rs150025731 and rs9913911 from a biological sample. It provides a method for determining single nucleotide polymorphisms (SNPs).

Another object of the present invention is to provide a method for treating open angle glaucoma in Koreans, comprising at least one single nucleotide polymorphism (SNP) selected from the group consisting of rs2946370, rs66500121, rs4308305, rs9853115, rs28795989, rs61275591, rs57111852, rs2745572, rs2526101, rs1412829, rs2472494, rs3829849, rs61870251, rs11024102, rs4303192, rs58073046, rs35155027, rs150025731, and rs9913911. To provide a biomarker composition for discrimination.

Another object of the present invention is to provide a composition for predicting the risk of developing open angle 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 open angle 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.

The present invention provides information necessary for predicting the risk of developing open angle glaucoma in Koreans, and comprises at least one selected from the group consisting of rs2946370, rs66500121, rs4308305, rs9853115, rs28795989, rs61275591, rs57111852, rs2745572, rs2526101, rs1412829, rs2472494, rs3829849, rs61870251, rs11024102, rs4303192, rs58073046, rs35155027, rs150025731 and rs9913911 from a biological sample. Provides a method for determining single nucleotide polymorphisms (SNPs).

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 rs2946370. 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, rs2946370, rs66500121, rs4308305, rs9853115, rs28795989, rs61275591, rs57111852, rs2745572, rs2526101, rs1412829, rs2472494, rs3829849, rs61870251, rs11024102, rs4303192, rs58073046, rs35155027, rs150025731 and rs9913911 used for predicting the risk of developing open-angle glaucoma in Koreans are from the National Center for Biotechnology Information (NCBI) single nucleotide polymorphism database (dbSNP, Each is registered at http:/http:/www.ncbi.nlm.nih.gov/snp/.

According to one embodiment of the present invention, in the case of rs2946370C>T, rs66500121G>A, rs4308305C>T, rs9853115A>T, rs28795989G>A, rs61275591G>A, rs2745572G>A, rs1412829G>A, rs2472494C>T, rs11024102C>T, rs4303192C>T, rs35155027C>G, rs150025731A>ACTCACCGG and single nucleotide polymorphism (SNP), it can be determined that the risk of open angle glaucoma is increased, and rs57111852A>G, In the case of single nucleotide polymorphisms (SNPs) rs2526101G>A, rs3829849C>T, rs61870251C>A, rs58073046G>A, rs9913911G>A, it can be determined that the risk of open angle glaucoma is reduced. In this regard, there was also a case where the patient with the highest genetic risk increased the risk of glaucoma by 5.62 times compared to the median.

In the present invention, the single nucleotide polymorphism (SNP) is located in the MIR4778 gene on chromosome 2 in the case of rs2946370, in the CADM2 gene on chromosome 3 in the case of rs66500121, in the FNDC3B gene on chromosome 3 in the case of rs4308305, in the LINC02020 gene on chromosome 3 in the case of rs9853115, in the AFAP1 gene on chromosome 4 in the case of rs28795989, in the C5orf67 gene on chromosome 5 in the case of rs61275591, in the EXOC2 gene on chromosome 6 in the case of rs57111852, in the FOXC1 gene on chromosome 6 in the case of rs2745572, in the THSD7A gene on chromosome 7 in the case of rs2526101, For rs1412829, it is located in the CDKN2B-AS1 gene on chromosome 9, for rs2472494, it is located in the ABCA1 gene on chromosome 9, for rs3829849, it is located in the LMX1B gene on chromosome 9, for rs61870251, it is located in the LHPP gene on chromosome 10, for rs11024102, it is located in the PLEKHA7 gene on chromosome 11, for rs4303192, it is located in the AGBL2 gene on chromosome 11, for rs58073046, it is located in the ARHGEF12 gene on chromosome 11, for rs35155027, it is located in the SIX1 gene on chromosome 14, for rs150025731, it is located in the LOXL1 gene on chromosome 15, and for rs9913911, it is located in the 17th It may be located in the GAS7 gene on the chromosome.

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 relates to a method for treating open angle glaucoma in Koreans, comprising at least one single nucleotide polymorphism (SNP) selected from the group consisting of rs2946370, rs66500121, rs4308305, rs9853115, rs28795989, rs61275591, rs57111852, rs2745572, rs2526101, rs1412829, rs2472494, rs3829849, rs61870251, rs11024102, rs4303192, rs58073046, rs35155027, rs150025731, and rs9913911. A biomarker composition for discrimination is provided.

In addition, the present invention provides a composition for predicting the risk of developing open angle 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 open angle 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 open-angle glaucoma in Koreans based on single nucleotide polymorphisms.

Figure 1 shows a histogram of PRS divided by the median.

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

The study population was extracted from the KoGES (Korean Genome and Epidemiology Study) urban cohort (HEXA cohort, Health examinee cohort). The KoGES database managed by the Korea Disease Control and Prevention Agency currently has 58,700 individuals genotyped using the Korean-chip microarray, Axiom Biobank version 1.1, and phenotype information was also provided to the Seoul National University College of Medicine Genome Medicine Research Institute (Director: Professor Kim Jong-il). Since all participant information in the KoGES DB is anonymized, individuals cannot be traced or identified with phenotypic and genotypic information, so there are no ethical issues and it is suitable for group research. Since KoGES participants were surveyed through a questionnaire about the presence or absence of glaucoma, this information was used to determine the patient group and the control group, and the genotype information was used to extract genotypes related to 19 SNPs and to calculate a polygenic risk score. Among the total study population of 58,600 people with a mean age of 53.8±8.0 years, 377 responded to the questionnaire that they had a history of glaucoma or treatment history.

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). In KoGES DB, DNA was extracted from blood to extract genotyping information, and genotyping was performed using Microarray sequencing (Korean Biobank Array version 1.1, Korean-chip). In this study, 19 SNPs (19 types including rs2946370, see Table 1) of each genetic locus for 19 species including MIR4778 were analyzed.

Genotyping for 19 polymorphisms including rs2946370 was performed with the 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 (Applied Biosystems, Foster City, CA), respectively.

1.3 Extraction of independent single nucleotide polymorphism (SNP) factors

Following three previous studies (Nature Communications, 2021; Gharahkhani/Nature Genetics, 2020; Craig/Nature Genetics, 2021; Sakaue), we extracted 19 independent SNPs with genome-wide significance from a genome-wide association study (GWAS) of glaucoma. Table 1 shows the 19 SNPs used in the construction of the PRS of the present invention.

No. rsID CHR POS(hg19) REF ALT OR BETA MAF P-value GENE (hg19) 1 rs2946370 2 66592411 C T 1.13 0.123 0.328 1.37.E-08 MIR4778Chr2:66,585,381-
66,585,460
(sequence number 1) 2 rs66500121 3 85137683 G A 1.12 0.113 0.174 2.30E-14 CADM2Chr3:85,008,140-
86,123,579
(sequence number 2) 3 rs4308305 3 171831116 C T 1.12 0.113 0.240 1.98E-09 FNDC3BChr3:171,757,368-
172,119,459
(sequence number 3) 4 rs9853115 3 186131600 A T 1.11 0.104 0.292 1.20.E-15 LINC02020Chr3:186,158,134-
186,163,031
(sequence number 4) 5 rs28795989 4 7891545 G A 1.15 0.140 0.180 2.80.E-25 AFAP1Chr4:7,760,440-
7,941,588
(sequence number 5) 6 rs61275591 5 55775556 G A 1.18 0.163 0.265 5.96.E-14 C5orf67Chr5:55,807,109-
55,902,083
(sequence number 6) 7 rs57111852 6 606263 A G 0.87 -0.139 0.185 8.50.E-13 EXOC2Chr6:485,154-
693,139
(sequence number 7) 8 rs2745572 6 1548369 G A 1.12 0.113 0.486 6.40.E-21 FOXC1Chr6:1,610,150-
1,614,132
(sequence number 8) 9 rs2526101 7 11677452 G A 0.90 -0.105 0.264 3.70.E-13 THSD7AChr7:11,409,992-
11,871,824
(sequence number 9) 10 rs1412829 9 22043926 G A 1.39 0.333 0.134 1.44E-27 CDKN2B-AS1Chr9:21,994,790-
22,121,096
(sequence number 10) 11 rs2472494 9 107695539 C T 1.20 0.185 0.457 1.11E-19 ABCA1Chr9:107,543,287-
107,690,436
(Sequence number 11) 12 rs3829849 9 129390800 C T 0.84 -0.174 0.078 6.03E-09 LMX1BChr9:129,376,207-
129,463,311
(Sequence number 12) 13 rs61870251 10 126284415 C A 0.87 -0.145 0.208 2.30E-10 LHPPChr10:126,150,392-
126,302,710
(Sequence number 13) 14 rs11024102 11 17008605 C T 1.10 0.094 0.465 9.99E-09 PLEKHA7Chr11:16,798,844-
17,035,961
(Sequence number 14) 15 rs4303192 11 47728924 C T 1.13 0.123 0.387 9.23E-09 AGBL2Chr11:47,681,143-
47,736,921
(Sequence number 15) 16 rs58073046 11 120248493 G A 0.88 -0.128 0.170 1.30.E-14 ARHGEF12Chr11:120,207,122-
120,360,646
(Sequence number 16) 17 rs35155027 14 61095174 C G 1.12 0.113 0.277 6.90.E-33 SIX1Chr14:61,110,139-
61,116,195
(Sequence number 17) 18 rs150025731 15 74223501 A ACTCACCGG 1.14 0.130 0.439 3.07E-15 LOXL1Chr15:74,218,803-
74,244,477
(Sequence number 18) 19 rs9913911 17 10031183 G A 0.91 -0.092 0.442 4.75E-08 GAS7Chr17:9,813,923-
10,101,923
(Sequence number 19)

We also extracted the odds ratio and minor allele frequency of each SNP. We applied the polygenic risk score (PRS) calculation formula to the KoGES city cohort (N=58,600) with the open-angle glaucoma questionnaire information. We assumed perfect linkage equilibrium for distant SNPs, meaning that these SNPs are completely independent of each other. For each individual in the KoGES database, we calculated a polygenic risk score (PRS), and observed the prevalence of open-angle glaucoma in each quintile of the PRS (ranked from lowest to highest) for validation, and also calculated the age of each participant.

Example 2. Experimental Results

The precision of the polygenic risk score was tested in quintiles of the PRS from 1 (low) to 5 (high). The PRS score divided by the median of the overall PRS ranged from 0.16 to 5.61 (Fig. 1).

The PRS results based on quintiles (N in CA indicates the number of patient cases) are shown in Table 2.

GRS5bin PRS average Number of cases Case frequency Total number 1 2.07096289 53 0.45221843 11720 2 2.860588022 62 0.529010239 11720 3 3.498454927 76 0.648464164 11720 4 4.262439959 73 0.622866894 11720 5 5.890287572 113 0.964163823 11720

GRS: Genetic risk score

PRS: Polygenc risk score

A total of 377 individuals were diagnosed with pre-existing open-angle glaucoma. The number of patients for each PRS quintile ranged from low to high PRS, 53, 62, 76, 73, and 113, respectively (Figure 2).

GRS10bin GRS average Number of cases Case frequency Total number 1 1.79609 23 0.392491 5860 2 2.345836 30 0.511945 5860 3 2.701569 27 0.460751 5860 4 3.019608 35 0.59727 5860 5 3.332795 38 0.648464 5860 6 3.664115 38 0.648464 5860 7 4.035477 33 0.56314 5860 8 4.489403 40 0.682594 5860 9 5.119029 47 0.802048 5860 10 6.661546 66 1.12628 5860

Meanwhile, the PRS results based on decile are shown in Table 3. A similar pattern of increase was derived from the PRS decile (Fig. 3). This provided additional validation for the newly established PRS. Among the glaucoma patients, the mean age of the PRS decile 1 (lowest PRS) was 60.7 years, and the mean age of the PRS decile 5 (highest PRS) was 59.3 years.

Accordingly, a polygenic risk score was derived based on 19 SNPs and the function of the PRS was verified in the KoGES cohort, confirming that the PRS of the present invention can be useful in determining the genetic risk of glaucoma.

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 (7)

In order to provide information necessary for predicting the risk of developing open angle glaucoma in Koreans, one or more of the following genes were selected from biological samples: rs2946370, rs66500121, rs4308305, rs9853115, rs28795989, rs61275591, rs57111852, rs2745572, rs2526101, rs1412829, rs2472494, rs3829849, rs61870251, rs11024102, rs4303192, rs58073046, rs35155027, rs150025731, and rs9913911. A method for determining single nucleotide polymorphisms (SNPs). In the first paragraph, The above single nucleotide polymorphisms (SNPs) are located in the MIR4778 gene on chromosome 2 for rs2946370, in the CADM2 gene on chromosome 3 for rs66500121, in the FNDC3B gene on chromosome 3 for rs4308305, in the LINC02020 gene on chromosome 3 for rs9853115, in the AFAP1 gene on chromosome 4 for rs28795989, in the C5orf67 gene on chromosome 5 for rs61275591, in the EXOC2 gene on chromosome 6 for rs57111852, in the FOXC1 gene on chromosome 6 for rs2745572, and in the THSD7A gene on chromosome 7 for rs2526101. For rs1412829, it is located in the CDKN2B-AS1 gene on chromosome 9, for rs2472494, it is located in the ABCA1 gene on chromosome 9, for rs3829849, it is located in the LMX1B gene on chromosome 9, for rs61870251, it is located in the LHPP gene on chromosome 10, for rs11024102, it is located in the PLEKHA7 gene on chromosome 11, for rs4303192, it is located in the AGBL2 gene on chromosome 11, for rs58073046, it is located in the ARHGEF12 gene on chromosome 11, for rs35155027, it is located in the SIX1 gene on chromosome 14, for rs150025731, it is located in the LOXL1 gene on chromosome 15, and for rs9913911, it is located in the 17th A method characterized by being located in the GAS7 gene of a chromosome. 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 biological sample is Korean. For the determination of open angle glaucoma in Koreans, comprising at least one single nucleotide polymorphism (SNP) selected from the group consisting of rs2946370, rs66500121, rs4308305, rs9853115, rs28795989, rs61275591, rs57111852, rs2745572, rs2526101, rs1412829, rs2472494, rs3829849, rs61870251, rs11024102, rs4303192, rs58073046, rs35155027, rs150025731, and rs9913911 Biomarker composition. A composition for predicting the risk of developing open angle glaucoma, comprising a preparation capable of detecting the biomarker composition of claim 5. A kit for predicting the risk of developing open angle glaucoma, comprising the composition of claim 6.

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