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

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

A method for determining the risk of developing open-angle glaucoma in Koreans using a polygenic risk score analysis.

[0001] 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.

[0002] Glaucoma is not caused by everyday lifestyle factors, is not contagious, and is not life-threatening. However, along with diabetic retinopathy, it is the most common cause of blindness in the eye. Because glaucoma, once it develops, damage to the optic nerve cannot be repaired, early diagnosis and treatment are crucial. Many ophthalmologists are currently researching the causes and effective treatments, but the exact pathogenesis of glaucoma remains unknown.

[0003] Specifically, glaucoma is a degenerative and progressive optic neuropathy characterized by glaucomatous optic nerve damage accompanied by visual field defects. Glaucoma is a leading cause of visual field loss and blindness worldwide. Recent studies have revealed that open-angle glaucoma (OAG) is phenotypically and genetically heterogeneous and is essentially polygenic.

[0004] 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 genome-wide association study (GWAS) studies on open-angle glaucoma is insufficient in East Asian races with a high incidence of glaucoma.

[0005] 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.

[0006] The purpose of the present invention is to provide information necessary for predicting the risk of developing open angle glaucoma in Koreans, and to identify at least one 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).

[0007] Another object of the present invention is to provide a method for determining 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.

[0008] 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.

[0009] Another object of the present invention is to provide a kit for predicting the risk of developing open angle glaucoma, comprising the composition.

[0010] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0011] 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).

[0012] In the present invention, polymorphism refers to the case where two or more alleles exist at a single locus, and among the polymorphic sites, a single nucleotide polymorphism (SNP) that differs only by a single base from person to person is called a single nucleotide polymorphism. 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 for a polymorphic marker means that there is an association for one or more specific alleles of a specific polymorphic marker. The marker may include any variant form of allele found in the genome, including single nucleotide polymorphisms (SNPs), microsatellites, insertions, deletions, duplications, and translocations.

[0013] In the present invention, an allele or biallelic trait refers to multiple types of a gene existing at the same locus on a homologous chromosome. The term allele is also used to indicate polymorphism; for example, a single nucleotide polymorphism (SNP) has two types of alleles.

[0014] In the present invention, "rs_id" refers to an independent marker, rs-ID, 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 rs2946370. The rs_id described in this table refers to the SNP marker, which is a polymorphic marker of the present invention. Those skilled in the art will be able to easily identify the location and sequence of the SNP using the rs_id.

[0015] 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 to predict the risk of developing open-angle glaucoma in Koreans are from the single nucleotide polymorphism database of the National Center for Biotechnology Information (NCBI dbSNP, Each is registered at http: / http: / www.ncbi.nlm.nih.gov / snp / .

[0016] 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 polymorphisms (SNPs), it can be determined that the risk for open angle glaucoma is increased, and rs57111852A>G, For the single nucleotide polymorphisms (SNPs) rs2526101G>A, rs3829849C>T, rs61870251C>A, rs58073046G>A, and rs9913911G>A, a reduced risk of open-angle glaucoma was identified. In this regard, patients with the highest genetic risk also had a 5.62-fold increased risk of glaucoma compared to the median.

[0017] 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 rs9853115, in the LINC02020 gene on chromosome 3 in the case of rs28795989, in the AFAP1 gene on chromosome 4 in the case of rs61275591, in the C5orf67 gene on chromosome 5 in the case of rs57111852, in the EXOC2 gene on chromosome 6 in the case of rs2745572, in the FOXC1 gene on chromosome 6 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 chromosome. It may be located in the GAS7 gene on the chromosome.

[0018] In the present invention, the biological sample may be blood, tissue, cells, serum, plasma, saliva, urine, etc., but is not limited thereto.

[0019] In the present invention, the subject of the biological sample may be a Korean.

[0020] In addition, the present invention provides a method for determining 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 is provided.

[0021] 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.

[0022] 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 including a SNP marker or a complementary polynucleotide thereof.

[0023] In the present invention, the primer or probe that specifically binds to the polynucleotide or its complementary polynucleotide is allele-specific.

[0024] In the present invention, a primer refers to a short nucleic acid sequence with a short free hydroxyl group that 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.

[0025] 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 as to confirm the presence or absence of a specific mRNA. 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.

[0026] 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).

[0027] In the present invention, the probe is an allele-specific probe, and due to the presence of a polymorphic site in the nucleic acid fragment, the probe may hybridize to a nucleic acid fragment containing one allele but not to a nucleic acid fragment containing the other allele. In this case, the hybridization conditions must be sufficiently stringent to ensure a significant difference in hybridization intensity between the alleles and to hybridize only to one of the alleles. Preferably, the probe may be single-stranded to maximize hybridization efficiency, but is not limited thereto.

[0028] In the present invention, detection of a specific nucleic acid using the primer can be performed by amplifying the sequence of the target gene using an amplification method such as PCR, and then confirming whether the gene has been amplified using a method known in the art. Furthermore, 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 a hybridized nucleic acid.

[0029] 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 (RNase mismatch cleavage, Myers et al., Science, 230, 1242-1246, 1985), single strand conformational polymorphism (single strand conformational polymorphism, Orita et al., Pro Nat Acad 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 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.

[0030] In addition, the present invention provides a kit for predicting the risk of developing open angle glaucoma, comprising the composition.

[0031] 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.

[0032] In the present invention, the kit may be a kit containing 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.

[0033] 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.

[0034] Figure 1 shows a histogram of PRS divided by the median.

[0035] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0036]

[0037] Example 1. Experimental method

[0038]

[0039] 1.1 Study group selection

[0040]

[0041] The study population was drawn from the KoGES (Korean Genome and Epidemiology Study) urban cohort (HEXA cohort, Health examinee cohort). Currently, all 58,700 individuals in the KoGES database, managed by the Korea Disease Control and Prevention Agency (KDCA), have been genotyped using the Korean-chip microarray (Axiom Biobank version 1.1). The phenotype information was also provided to the Genomic Medicine Research Institute (Director: Professor Kim Jong-il) at Seoul National University College of Medicine. The KoGES database has the advantage of being ethically sound, as all participant information is anonymized, and phenotypic and genotypic information cannot be used to trace or identify individuals, making it suitable for conducting population-based studies. KoGES participants were surveyed through a questionnaire regarding the presence or absence of glaucoma, which was used to distinguish between case and control groups. The genotype information was used to derive a polygenic risk score by extracting genotypes associated with 19 single nucleotide polymorphisms (SNPs). Among the 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.

[0042]

[0043] 1.2 Sample preparation and allele determination

[0044]

[0045] Genomic DNA was isolated from blood using the MG Blood Genomic DNA Extraction SV kit (MGmed, Seoul, Korea). To extract genotypic information from the KoGES database, DNA was extracted from blood and genotyped using microarray sequencing (Korean Biobank Array version 1.1, Korean-chip). Nineteen SNPs (including rs2946370, see Table 1) at each of the 19 loci for 19 species, including MIR4778, were analyzed in this study.

[0046] Genotyping for 19 polymorphisms including rs2946370 was performed with a QuantStudio real-time polymerase chain reaction system (Applied Biosystems) using TaqMan SNP Genotyping Assay assay IDs of C___2485201_10, C__11916555_30, C__15840320_10, and C___2485225_20 (Applied Biosystems, Foster City, CA), respectively.

[0047]

[0048] 1.3 Extraction of independent single nucleotide polymorphism (SNP) factors

[0049]

[0050] 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.

[0051]

[0052] No.rsIDCHRPOS(hg19)REFALTORBETAMAFP-valueGENE (hg19)1rs2946370266592411CT1.130.1230.3281.37.E-08MIR4778Chr2:66,585,381-66,585,460(SEQ ID NO: 1)2rs66500121385137683GA1.120.1130.1742.30E-14CADM2Chr3:85,008,140-86,123,579(SEQ ID NO: 2) 3rs43083053171831116CT1.120.1130.2401.98E-09FNDC3BChr3:171,757,368-172,119,459 (SEQ ID NO: 3) 4rs98531153186131600AT1.110.1040.2921.20.E-15LINC02020Chr3:186,158,134-186,163,031 (SEQ ID NO: 4) 5rs2879598947891545GA1.150.1400.1802.80.E-25AFAP1Chr4:7,760,440-7,941,588 (SEQ ID NO: 5) 6rs61275591555775556GA1.180.1630.2655.96.E-14C5orf67Chr5:55,807,109-55,902,083 (SEQ ID NO: 6) 7rs571118526606263AG0.87-0.1390.1858.50.E-13EXOC2Chr6:485,154-693,139 (SEQ ID NO: 7) 8rs274557261548369GA1.120.1130.4866.40.E-21FOXC1Chr6:1,610,150-1,614,132 (SEQ ID NO: 8)9rs2526101711677452GA0.90-0.1050.2643.70.E-13THSD7AChr7:11,409,992-11,871,824 (SEQ ID NO: 9)10rs1412829922043926GA1.390.3330.1341.44E-27CDKN2B-AS1Chr9:21,994,790-22,121,096 (SEQ ID NO: 10)11rs24724949107695539CT1.200.1850.4571.11E-19ABCA1Chr9:107,543,287-107,690,436(SEQ ID NO: 11)12rs38298499129390800CT0.84-0.1740.0786.03E-09LMX1BChr9:129,376,207-129,463,311(SEQ ID NO: 12)13rs6187025110126284415CA0.87-0.1450.2082.30E-10LHPPChr10:126,150,392-126,302,710(SEQ ID NO: 13)14rs110241021117008605CT1.100.0940.4659.99E-09PLEKHA7Chr11:16,798,844-17,035,961 (SEQ ID NO: 14)15rs43031921147728924CT1.130.1230.3879.23E-09AGBL2Chr11:47,681,143-47,736,921 (SEQ ID NO: 15)16rs5807304611120248493GA0.88-0.1280.1701.30.E-14ARHGEF12Chr11:120,207,122-120,360,646 (SEQ ID NO: 16)17rs351550271461095174CG1.120.1130.2776.90.E-33SIX1Chr14:61,110,139-61,116,195 (SEQ ID NO: 17)18rs1500257311574223501AACTCACCGG1.140.1300.4393.07E-15LOXL1Chr15:74,218,803-74,244,477 (SEQ ID NO: 18)19rs99139111710031183GA0.91-0.0920.4424.75E-08GAS7Chr17:9,813,923-10,101,923 (SEQ ID NO: 19).

[0053]

[0054] 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) using open-angle glaucoma questionnaire information. We assumed perfect linkage equilibrium for distant SNPs, meaning that these SNPs were completely independent of each other. We calculated a polygenic risk score (PRS) for each individual in the KoGES database, and for validation, we observed the prevalence of open-angle glaucoma in each quintile of the PRS (ranked from lowest to highest). We also calculated the age of each participant.

[0055]

[0056] Example 2. Experimental Results

[0057]

[0058] The precision of the polygenic risk score was tested using quintiles of the PRS ranging 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).

[0059] The PRS results based on quintiles (N in CA indicates the number of patient cases) are shown in Table 2.

[0060]

[0061] GRS5binPRS Average Cases Cases Frequency Total Number 12.07096289530.452218431172022.860588022620.5290102391172033.498454927760.6484641641172044.262439959730.6228668941172055.8902875721130.96416382311720

[0062] GRS: Genetic risk score

[0063] PRS: Polygenic risk score

[0064]

[0065] A total of 377 individuals were diagnosed with pre-existing open-angle glaucoma. The number of patients in each PRS quintile ranged from low to high PRS, 53, 62, 76, 73, and 113, respectively (Fig. 2).

[0066]

[0067] GRS10binGRS Average Cases Investigated Cases Frequency Total Number 11.79609230.392491586022.345836300.511945586032.701569270.460751586043.019608350.59727586053.332795380.648464586063.664115380.648464586074.035477330.56314586084.489403400.682594586095.119029470.8020485860106.661546661.126285860

[0068]

[0069] Meanwhile, the PRS results based on decile scores are presented in Table 3. A similar pattern of increase was observed across the decile scores (Fig. 3). This provided further validation of the newly established PRS. Among glaucoma patients, the mean age in the 1st decile of the PRS (lowest PRS) was 60.7 years, and the mean age in the 5th decile of the PRS (highest PRS) was 59.3 years.

[0070] 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.

[0071]

[0072] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. 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 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 biological samples. How to determine single nucleotide polymorphisms (SNPs).

2. In paragraph 1, 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 chromosome. A method characterized in that it is located in the GAS7 gene of a chromosome.

3. In paragraph 1, 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.

4. In paragraph 1, A method characterized in that the subject of the above biological sample is Korean.

5. A biomarker for the detection 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. Composition.

6. A composition for predicting the risk of developing open angle glaucoma, comprising a preparation capable of detecting the biomarker composition of clause 5.

7. A kit for predicting the risk of developing open angle glaucoma, comprising the composition of Article 6.

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