JP6998476B2 - How to determine the risk of developing myopia - Google Patents

Description

The present invention relates to a method for determining the risk of developing myopia using a single nucleotide polymorphism (SNP) having a high genetic effect.

Myopia is defined as having an equivalent spherical power (SE) of -0.5 D (diopter) or higher, and is the most common refractive error in the world. Severe myopia is commonly defined as SE <-6.0 D or axial length> 26.0 mm and can cause a variety of retinal detachments, glaucoma, cataracts, macular bleeding, macular degeneration, etc. that can cause blindness and severe visual impairment. Increases the risk of developing eye diseases and systemic diseases (Non-Patent Documents 1 and 2). Myopia is more common in the Asian population than in the non-Asian population (Non-Patent Document 3-8), and the prevalence of severe myopia in the young adult population is higher in the Asian region (2.0-2.3%) than in non-Asian countries (2.0-2.3%). It has been reported to be extremely high at 6.8-21.6%) (Non-Patent Document 9). The prevalence of myopia has continued to rise dramatically worldwide over the last few decades (Non-Patent Document 10), and recent reports indicate that by 2050, the number of myopia patients worldwide will be approximately 5 billion. It is predicted that the number of people (almost half of the world's population) and the number of patients with severe myopia will reach about 1 billion (about 10% of the world's population) (Non-Patent Document 11). Although the etiology of myopia has not been elucidated accurately, myopia is considered to be a multifactorial disease that develops due to the combined involvement of various environmental and genetic factors (Non-Patent Document 12-17). It is suggested that the higher the intensity of myopia, the stronger the contribution of genetic factors (Non-Patent Documents 18-20).

Genome-wide association study (GWAS) is a method for searching for gene polymorphisms that cover the entire genome and show a significant frequency difference between unrelated patient populations and healthy populations. It is very effective in elucidating the genetic factors of factor diseases. Since 2009, multiple GWAS for myopia have been carried out, and to date, a large number of susceptibility gene regions for myopia and myopia-related traits have been identified in various racial populations (Non-Patent Document 21-24). .. The NHGRI-EBI GWAS Catalog (https://www.ebi.ac.uk/gwas/), a database that summarizes the results of GWAS studies reported as a paper, contains about candidate gene regions for myopia and myopia-related traits. It has been reported in 200 places (Non-Patent Document 25). However, it is estimated that these approximately 200 candidate gene regions are only part of the overall genetic factors of myopia and myopia-related traits. Two 2013 papers on large-scale GWAS on myopia and refractive error reported more than 30 susceptibility gene regions (Non-Patent Documents 26 and 27), but these gene regions are phenotypic dispersions of myopia and refractive error. It is estimated to be less than 12% of the total (Non-Patent Document 17). Moreover, in many of the approximately 200 candidate gene regions, the strength or presence or absence of correlation between myopia or myopia-related traits and the candidate gene region varies between racial populations or the severity of myopia. Is recognized. These facts imply that there are many unidentified genetic factors that contribute to myopia / myopia-related traits.

Saw SM, Gazzard G, Shih-Yen EC, Chua WH. Myopia and associated pathological complications. Ophthalmic Physiol Opt. 2005; 25 (5): 381-391. Burton TC. The influence of refractive error and lattice degeneration on the incidence of retinal detachment. Trans Am Ophthalmol Soc. 1989; 87: 143-157. Wilson A, Woo G. A review of the prevalence and causes of myopia. Singapore Med J. 1989; 30 (5): 479-484. Lam CS, Goldschmidt E, Edwards MH. Prevalence of myopia in local and international schools in Hong Kong. Optom Vis Sci. 2004; 81 (5): 317-322. Kempen JH, Mitchell P, Lee KE, Tielsch JM, Broman AT, Taylor HR, et al. The prevalence of refractive errors among adults in the United States, Western Europe, and Australia. Arch Ophthalmol. 2004; 122 (4): 495 -505. Sawada A, Tomidokoro A, Araie M, Iwase A, Yamamoto T; Tajimi Study Group. Refractive errors in an elderly Japanese population: the Tajimi study. Ophthalmology. 2008; 115 (2): 363-370. Pan CW, Klein BE, Cotch MF, Shrager S, Klein R, Folsom A, et al. Racial variations in the prevalence of refractive errors in the United States: the multi-ethnic study of atherosclerosis. Am J Ophthalmol. 2013; 155 ( 6): 1129-1138. Pan CW, Zheng YF, Anuar AR, Chew M, Gazzard G, Aung T, et al. Prevalence of refractive errors in a multiethnic Asian population: the Singapore epidemiology of eye disease study. Invest Ophthalmol Vis Sci. 2013; 54 (4) : 2590-2598. Wong YL, Saw SM. Epidemiology of Pathologic Myopia in Asia and Worldwide. Asia Pac J Ophthalmol (Phila). 2016; 5 (6): 394-402. Dolgin E. The myopia boom. Nature. 2015; 519 (7543): 276-278. Holden BA, Fricke TR, Wilson DA, Jong M, Naidoo KS, Sankaridurg P, et al. Global Prevalence of Myopia and High Myopia and Temporal Trends from 2000 through 2050. Ophthalmology. 2016; 123 (5): 1036-1042. Baird PN, Schache M, Dirani M. The GEnes in Myopia (GEM) study in understanding the aetiology of refractive errors. Prog Retin Eye Res. 2010; 29 (6): 520-542. Au Eong KG, Tay TH, Lim MK. Education and myopia in 110,236 young Singaporean males. Singapore Med J. 1993; 34 (6): 489-492. Au Eong KG, Tay TH, Lim MK. Race, culture and myopia in 110,236 young Singaporean males. Singapore Med J. 1993; 34 (1): 29-32. Morgan IG, Rose KA. Myopia and international academic performance. Ophthalmic Physiolog Opt. 2013; 33 (3): 329-338. French AN, Ashby RS, Morgan IG, Rose KA. Time outdoors and the prevention of myopia. Exp Eye Res. 2013; 114: 56-68. Fan Q, Verhoeven VJ, Wojciechowski R, Barathi VA, Hysi PG, Guggenheim JA, et al. Meta-analysis of gene-environment-wide association scans accounting for education level identifies additional loci for refractive error. Nat Commun. 2016; 7: 11008. Guggenheim JA, Kirov G, Hodson SA. The heritability of high myopia: a reanalysis of Goldschmidt's data. J Med Genet. 2000; 37 (3): 227-231. Farbrother JE, Kirov G, Owen MJ, Guggenheim JA. Family aggregation of high myopia: estimation of the sibling recurrence risk ratio. Invest Ophthalmol Vis Sci. 2004; 45 (9): 2873-2878. Peet JA, Cotch MF, Wojciechowski R, Bailey-Wilson JE, Stambolian D. Heritability and familial aggregation of refractive error in the Old Order Amish. Invest Ophthalmol Vis Sci. 2007; 48 (9): 4002-4006. Li J, Zhang Q. Insight into the molecular genetics of myopia. Mol Vis. 2017; 23: 1048-1080. Fan Q, Barathi VA, Cheng CY, Zhou X, Meguro A, Nakata I, et al. Genetic variants on chromosome 1q41 influence ocular axial length and high myopia. PLoS Genet. 2012; 8 (6): e1002753. Solouki AM, Verhoeven VJ, van Duijn CM, Verkerk AJ, Ikram MK, Hysi PG, et al. A genome-wide association study identifies a susceptibility locus for refractive errors and myopia at 15q14. Nat Genet. 2010; 42 (10): 897-901. Hysi PG, Young TL, Mackey DA, Andrew T, Fernandez-Medarde A, Solouki AM, et al. A genome-wide association study for myopia and refractive error identifies a susceptibility locus at 15q25. Nat Genet. 2010; 42 (10) : 902-905. MacArthur J, Bowler E, Cerezo M, Gil L, Hall P, Hastings E, et al. The new NHGRI-EBI Catalog of published genome-wide association studies (GWAS Catalog). Nucleic Acids Res. 2017; 45 (D1): D896-D901. Kiefer AK, Tung JY, Do CB, Hinds DA, Mountain JL, Francke U, et al. Genome-wide analysis points to roles for extracellular matrix remodeling, the visual cycle, and neuronal development in myopia. PLoS Genet. 2013; 9 ( 2): e1003299. Verhoeven VJ, Hysi PG, Wojciechowski R, Fan Q, Guggenheim JA, Hohn R, et al. Genome-wide meta-analyses of multiancestry cohorts identify multiple new susceptibility loci for refractive error and myopia. Nat Genet. 2013; 45 (3) : 314-318.

As mentioned above, myopia is considered to be a disease that develops due to the combined involvement of genetic factors and environmental factors (Non-Patent Documents 12 to 17), and the higher the intensity of myopia, the stronger the genetic factors. It is suggested that it contributes (Non-Patent Documents 18 to 20). The previously reported gene polymorphisms that correlate with the risk of developing myopia are gene polymorphisms identified by genetic studies mainly targeting myopia cases including weak myopia and moderate myopia, but they are weak. The group of cases with moderate myopia includes many cases that are greatly affected by environmental factors. Since it is hard to say that the gene polymorphism identified for such a case group has a high genetic effect on the onset of myopia, it is not always effective as a gene polymorphism used for determining the risk of developing myopia. Moreover, with such gene polymorphisms, it is difficult to accurately determine the risk of developing myopia in areas with a high prevalence of severe myopia. An object of the present invention is to provide a means for determining the risk of developing myopia more accurately than in the prior art in various regions and races including regions with a high prevalence of severe myopia.

The inventors of the present application obtained samples from a huge number of patients with severe myopia and a group of healthy subjects and conducted a diligent genetic analysis on Japanese subjects with a high prevalence of severe myopia. We have completed the invention of the present application by identifying nine single nucleotide polymorphisms (SNPs) that correlate with a wide range of significance.

That is, the present invention uses the following (1) to (9) single nucleotide polymorphisms (SNP): using genomic DNA samples isolated from human subjects.
(1) SNP of SNP ID number rs72748160 at position 85,692,651 (position 201 in SEQ ID NO: 1) of chromosome 15.
(2) SNP of SNP ID number rs74633073 at position 69,229,747 of chromosome 3 (position 201 in SEQ ID NO: 2)
(3) SNP of SNP ID number rs76903431 at position 130,041,322 (position 201 in SEQ ID NO: 3) of chromosome 12.
(4) SNP of SNP ID number rs2246661 at position 204,974,863 (position 201 in SEQ ID NO: 4) of chromosome 1.
(5) SNP of SNP ID number rs12032649 at position 219,605,617 of chromosome 1 (position 201 in SEQ ID NO: 5)
(6) SNP of SNP ID number rs698047 at position 41,867,932 of chromosome 1 (position 201 in SEQ ID NO: 6)
(7) SNP of SNP ID number rs17029206 at position 1,739,832 of chromosome 3 (position 201 in SEQ ID NO: 7)
(8) SNP of SNP ID number rs589135 at position 34,709,241 (position 201 in SEQ ID NO: 8) of chromosome 15.
(9) SNP of SNP ID number rs28415942 at position 79,092,508 of chromosome 15 (position 201 in SEQ ID NO: 9)
A method for determining the risk of developing myopia, which involves examining the genotypes of at least 5 SNPs selected from the above, and the risk allele that is an indicator of the high risk of developing myopia is (1) T. (2) is T, (3) is A, (4) is C, (5) is G, (6) is G, (7) is T, (8) ) Is G and (9) is T, providing a method.

INDUSTRIAL APPLICABILITY The present invention provides a means for determining the risk of developing myopia more accurately than in the prior art. An SNP that significantly correlates with the risk of developing myopia can be said to be an SNP with a high genetic effect on the risk of developing myopia. By using such SNP as an index, it is possible to more appropriately evaluate the genetic effect on the risk of developing myopia, and the risk of developing myopia is determined more accurately than in the prior art (determination of the risk of developing myopia by a doctor or the like). Can be assisted). In addition to genetic testing aimed at knowing the risk of developing myopia itself, the present invention is implemented for subjects before the onset of myopia, and lifestyle guidance (computers, smartphones, video games) to suppress the progression of myopia. , Advice on improving the living environment such as reading), orthokeratology, low-concentration atropine treatment, and other medical procedures that suppress the progression of myopia can be used at an early stage.

Table 1 shows the nine SNPs identified by the inventors of the present application that significantly correlate with intense myopia.

In addition, in this specification, the position on the chromosome of an arbitrary base is shown based on the human genome reference sequence GRCh38. SNP ID numbers starting with rs are references in dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/), the SNP database of the National Center for Biotechnology Information (NCBI). SNP ID number. A person skilled in the art can easily obtain information such as the physical position of the SNP on the chromosome and the base sequence in the vicinity thereof based on this SNP ID number. SEQ ID NOs: 1 to 9 in the sequence listing of the present application show the genomic nucleotide sequences of 200 bp before and after each SNP, but genomic nucleotide sequences adjacent to these regions can be easily obtained by those skilled in the art. ..

The SNPs shown in Table 1 were comprehensively identified as SNPs that significantly correlate with severe myopia by GWAS in the Japanese population of patients with severe myopia and the population of healthy subjects. However, the Japanese population was targeted at patients with severe myopia, whose prevalence of severe myopia in the Asian region including Japan was significantly higher than in other regions, and the contribution of genetic factors is considered to be particularly large. This is because a large number of samples can be obtained. The target race that can accurately determine the risk of developing myopia by the method of the present invention is not limited to Japanese, but it is also possible to determine people in Asian regions other than Japan, and in Europe, the United States and other non-Asian regions. Therefore, the subjects to which the method of the present invention can be implemented include Asians including Japanese and non-Asians such as Westerners. When the method of the present invention is utilized for suppressing the progression of myopia of a subject, a young subject (for example, about 15 years old or younger) may be the main target.

In the method of the present invention, at least one of the nine SNPs shown in Table 1, for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight. Examine the above or all 9 genotypes. Since the average number of risk alleles carried in 1,586 healthy subjects was 4.39 ± 1.21 (see the examples below), it is possible to examine the genotypes of at least 5, more preferably all 9 of them. desirable. When examining only a part of the nine SNPs, any SNP may be selected, but the one having a high odds ratio, that is, the SNP listed higher in Table 1 may be preferentially selected. For example, when investigating the genotypes of 5 or more SNPs among (1) to (9), it is preferable to select 5 or more SNPs including (1) to (5).

Further, instead of the SNPs (1) to (9), the genotypes of SNPs that are in linkage disequilibrium with each SNP may be investigated. As is well known in the art, when multiple SNPs are in a strong linkage disequilibrium, they behave similarly. For example, when SNP-A, SNP-B, and SNP-C are in a strong linkage disequilibrium, and SNP-A shows a significant correlation with disease X, SNP-B and SNP-C are also significant with disease X. Correspondence is shown. Therefore, if the SNP is in a strong linkage disequilibrium state with each of the SNPs (1) to (9), it significantly correlates with the intense myopia as in the SNPs of (1) to (9), so that the risk of developing myopia is determined. Can be used for.

Linkage disequilibrium coefficients such as D'and r ² are known as values representing the strength of linkage disequilibrium. D'and r ² are calculated by Gabriel et al. (Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, et al. The structure of haplotype blocks in the human genome. Science. 2002; 296 (5576) ): 2225-2229.). Generally, SNPs having a relationship of r ² > 0.7 are considered to be in a strong linkage disequilibrium state. In the present invention, the term "SNP in linkage disequilibrium" with a certain SNP means an SNP having a relationship of r ² > 0.7 with the SNP. Information on "SNPs in linkage disequilibrium (SNPs with a relationship of r ² >0.7)" with specific SNPs can be found, for example, in the National Cancer Institute (NCI) linkage disequilibrium database LDlink ( It can be easily searched by LDproxy (https://ldlink.nci.nih.gov/?tab=ldproxy) in https://ldlink.nci.nih.gov/).

In the present invention, when investigating the genotypes of a plurality of SNPs, for example, the subject is determined by the number of genotypes (high-risk genotypes) having risk alleles (allyls showing a positive correlation with the risk of developing severe myopia). The risk of developing myopia can be determined. In this case, it is shown that the higher the number of high-risk genotypes, the higher the risk of developing myopia. Since the average number of high-risk genotypes possessed by healthy subjects was 4.39 ± 1.21, "5 or more high-risk genotypes" exceeding this average value can be used as a guide to judge that the risk of developing myopia is high. good.

In addition, each risk allele may be weighted by odds ratio. Among subjects with two high-risk genotypes, subjects A with high-risk genotypes for (4) (rs2246661, odds ratio 1.31) and (6) (rs698047, odds ratio 1.28) Compared with subject B, who has a high-risk genotype for (1) (rs72748160, odds ratio 1.93) and (2) (rs74633073, odds ratio 1.91), subject B is better. It can be evaluated that the risk of developing myopia is higher. In the method of the present invention, in addition to the number of high-risk genotypes, the high odds ratio of detected high-risk genotypes (types and combinations of high-risk genotypes) is also taken into consideration to determine the risk of developing myopia. May be good. For example, the odds ratio or an appropriate numerical value according to the magnitude of the odds ratio is set as the risk allele score of each SNP, and the scores of the high-risk genotypes detected in the subject are added or integrated. A judgment method such as score evaluation of the subject's risk of developing myopia can be considered. The odds ratio values shown in Table 1 may be used, or the odds ratios may be calculated by proceeding with analysis for a large number of patients with severe myopia, and the obtained values may be replaced with the odds ratios in Table 1 for use. You may.

The genomic DNA sample used in the method of the present invention can be easily prepared by a conventional method from peripheral blood, oral mucosa, etc. collected from a subject.

The method itself for investigating the genotype of SNP is well known, and the method is not particularly limited in the present invention. For example, the base sequence of the SNP site may be determined by sequencing and the genotype may be examined. Further, as an SNP typing method, various methods such as an invader method, a TaqMan-PCR method, an Allele Specific Primer (ASP) method, and a typing method using a chip equipped with an SNP detection probe are known. All of them are conventional methods, and various kits containing the reagents required for these methods are also commercially available. When investigating the genotype of SNP in the present invention, any known method may be used. Primers, probes, etc. required for genotype detection can be easily designed and prepared by those skilled in the art based on the base sequence information in the vicinity of SNP.

The probe for SNP detection used in SNP typing methods such as the TaqMan-PCR method usually consists of the same base sequence as the base sequence of the genomic partial region containing several bases before and after the SNP to be tested, or a base sequence complementary thereto. It has a structure in which an additional nucleic acid portion such as a linker or an adapter is linked to a nucleic acid portion (a portion that hybridizes to a target genomic region), if necessary. The overall length of the part that hybridizes to the target genomic region (or the total length of the probe if no additional nucleic acid part is included) is approximately 15 to 30 bases, for example, 16 to 30 bases, 17 to 30 bases, or It is about 18 to 30 bases.

(1) The probe for detecting the genotype of rs72748160 is from a base sequence of about 15 to 30 bases including position 201 (SNP of rs72748160) of SEQ ID NO: 1 and several bases before and after it, or a base sequence complementary thereto. Nucleic acid moiety is included as a moiety that hybridizes to the target genomic region. For example, it may be a probe having the same base sequence as a partial region of about 15 to 30 bases including positions 197 to 205 in the base sequence shown in SEQ ID NO: 1, or a probe having a base sequence complementary thereto. In this case, the probe is TTAG [G] GCAA, TTAG [T] GCAA, TTGC [C] CTAA, or TTGC [A] CTAA as the sequence at positions 197 to 205 in SEQ ID NO: 1 or a sequence complementary thereto. ([] Is the SNP part) is included.

(2) The probe for detecting the genotype of rs74633073 is from a base sequence of about 15 to 30 bases including position 201 (SNP of rs74633073) of SEQ ID NO: 2 and several bases before and after it, or a base sequence complementary thereto. Nucleic acid moiety is included as a moiety that hybridizes to the target genomic region. For example, it may be a probe having the same base sequence as a partial region of about 15 to 30 bases including positions 197 to 205 in the base sequence shown in SEQ ID NO: 2, or a probe having a base sequence complementary thereto. In this case, the probe is CTGT [C] GCCA, CTGT [T] GCCA, TGGC [G] ACAG, or TGGC [A] ACAG as the sequence at positions 197 to 205 in SEQ ID NO: 2 or a sequence complementary thereto. ([] Is the SNP part) is included.

(3) The probe for detecting the genotype of rs76903431 is from a base sequence of about 15 to 30 bases including position 201 (SNP of rs76903431) of SEQ ID NO: 3 and several bases before and after it, or a base sequence complementary thereto. Nucleic acid moiety is included as a moiety that hybridizes to the target genomic region. For example, it may be a probe having the same base sequence as a partial region of about 15 to 30 bases including positions 197 to 205 in the base sequence shown in SEQ ID NO: 3, or a probe having a base sequence complementary thereto. The probe in this case is ATAG [A] TCAA, ATAG [T] TCAA, TTGA [T] CTAT, or TTGA [A] CTAT as the sequence at positions 197 to 205 in SEQ ID NO: 3 or a sequence complementary thereto. ([] Is the SNP part) is included.

(4) The probe for detecting the genotype of rs2246661 is from a base sequence of about 15 to 30 bases including position 201 (SNP of rs2246661) of SEQ ID NO: 4 and several bases before and after it, or a base sequence complementary thereto. Nucleic acid moiety is included as a moiety that hybridizes to the target genomic region. For example, it may be a probe having the same base sequence as a partial region of about 15 to 30 bases including positions 197 to 205 in the base sequence shown in SEQ ID NO: 4, or a probe having a base sequence complementary thereto. In this case, the probe is GGGA [C] AAGG, GGGA [T] AAGG, CCTT [G] TCCC, or CCTT [A] TCCC as the sequence at positions 197 to 205 in SEQ ID NO: 4 or a sequence complementary thereto. ([] Is the SNP part) is included.

(5) The probe for detecting the genotype of rs12032649 is from a base sequence of about 15 to 30 bases including position 201 (SNP of rs12032649) of SEQ ID NO: 5 and several bases before and after it, or a base sequence complementary thereto. Nucleic acid moiety is included as a moiety that hybridizes to the target genomic region. For example, it may be a probe having the same base sequence as a partial region of about 15 to 30 bases including positions 197 to 205 in the base sequence shown in SEQ ID NO: 5, or a probe having a base sequence complementary thereto. In this case, the probe is GCCA [G] CTGG, GCCA [T] CTGG, CCAG [C] TGGC, or CCAG [A] TGGC as the sequence at positions 197 to 205 in SEQ ID NO: 5 or a sequence complementary thereto. ([] Is the SNP part) is included.

(6) The probe for detecting the genotype of rs698047 is from a base sequence of about 15 to 30 bases including position 201 (SNP of rs698047) of SEQ ID NO: 6 and several bases before and after it, or a base sequence complementary thereto. Nucleic acid moiety is included as a moiety that hybridizes to the target genomic region. For example, it may be a probe having the same base sequence as a partial region of about 15 to 30 bases including positions 197 to 205 in the base sequence shown in SEQ ID NO: 6, or a probe having a base sequence complementary thereto. The probe in this case is CCCC [C] TCCC, CCCC [G] TCCC, GGGA [G] GGGG, or GGGA [C] GGGG as the sequence at positions 197 to 205 in SEQ ID NO: 6 or a sequence complementary thereto. ([] Is the SNP part) is included.

(7) The probe for detecting the genotype of rs17029206 is from a base sequence of about 15 to 30 bases including position 201 (SNP of rs17029206) of SEQ ID NO: 7 and several bases before and after it, or a base sequence complementary thereto. Nucleic acid moiety is included as a moiety that hybridizes to the target genomic region. For example, it may be a probe having the same base sequence as a partial region of about 15 to 30 bases including positions 197 to 205 in the base sequence shown in SEQ ID NO: 7, or a probe having a base sequence complementary thereto. The probe in this case is GAAA [C] TTAC, GAAA [T] TTAC, GTAA [G] TTTC, or GTAA [A] TTTC as the sequence at positions 197 to 205 in SEQ ID NO: 7 or a sequence complementary thereto. ([] Is the SNP part) is included.

(8) The probe for detecting the genotype of rs589135 is from a base sequence of about 15 to 30 bases including position 201 (SNP of rs589135) of SEQ ID NO: 8 and several bases before and after it, or a base sequence complementary thereto. Nucleic acid moiety is included as a moiety that hybridizes to the target genomic region. For example, it may be a probe having the same base sequence as a partial region of about 15 to 30 bases including positions 197 to 205 in the base sequence shown in SEQ ID NO: 8, or a probe having a base sequence complementary thereto. The probe in this case is GAAG [A] GGCT, GAAG [G] GGCT, AGCC [T] CTTC, or AGCC [C] CTTC as the sequence at positions 197 to 205 in SEQ ID NO: 8 or a sequence complementary thereto. ([] Is the SNP part) is included.

(9) The probe for detecting the genotype of rs28415942 is from a base sequence of about 15 to 30 bases including position 201 (SNP of rs28415942) of SEQ ID NO: 9 and several bases before and after it, or a base sequence complementary thereto. Nucleic acid moiety is included as a moiety that hybridizes to the target genomic region. For example, it may be a probe having the same base sequence as a partial region of about 15 to 30 bases including positions 197 to 205 in the base sequence shown in SEQ ID NO: 9, or a probe having a base sequence complementary thereto. The probe in this case is CACA [C] CCAC, CACA [T] CCAC, GTGG [G] TGTG, or GTGG [A] TGTG as the sequence at positions 197 to 205 in SEQ ID NO: 9 or a sequence complementary thereto. ([] Is the SNP part) is included.

The method for determining the risk of developing severe myopia according to the present invention may be carried out in combination with other genetic diagnosis methods. For example, genetic testing for various items using a microarray chip that combines a probe that detects polymorphic genotypes related to disease risk and constitution and a probe that detects SNPs shown in Table 1. The risk of developing severe myopia may be determined according to the present invention as one of the items.

According to the present invention, it is possible to provide information or advice that a subject determined to be at high risk has a high risk of developing myopia. By providing such information, advice on myopia prevention by improving the living environment (environment such as personal computer, smartphone, video game, reading, etc.) and suppression of myopia progression by orthokeratology or low-concentration atropine treatment, etc. It becomes possible to carry out preventive measures or preventive measures at an early stage.

Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to the following examples.

Materials and Methods Targets A total of 1,668 unrelated Japanese patients with severe myopia (at least one eye SE <-9.0 D) and unrelated Japanese healthy controls (both eyes -0.5 D <SE <+2.0 D) ) A total of 1,601 people were targeted for the GWAS discovery stage. Collection of patient cases and healthy subjects was carried out at Yokohama City University, Okada Ophthalmology and Aoto Ophthalmology in Yokohama City, Kanagawa Prefecture. All subjects were comprehensive ophthalmologic examinations including axial length (AL), fundus examination, spherical power, and corneal curvature (autorefractors are ARK-730A (Nidek), ARK-700A (Nidek), KP-8100P ( Topcon) was diagnosed with AL-2000 (Tome) as the biometer / pachymeter). Patients with severe myopia were confirmed to be free of hereditary disorders known to be associated with at least either myopia or severe myopia (glaucoma, keratoconus, Marfan syndrome, etc.). The QIAamp DNA Blood Maxi Kit (QIAGEN) was used to collect peripheral blood lymphocytes and extract genomic DNA from peripheral blood cells. Experimental procedures were performed under standard conditions to prevent variability in DNA quality.

For the replication stage (confirmation study), an unrelated Japanese population (500 patients with binocular AL> 26.0 mm and 4,869 healthy controls) different from the GWAS stage was added. Japanese cases of severe myopia were collected from the Kyoto High Myopia Cohort [1-3]. In addition, the general healthy subjects [4,5] derived from the Nagahama Proactive Genome Cohort (Nagahama Study) for the comprehensive human bioscience dataset, which were recruited from the general public living in Nagahama City, Shiga Prefecture from 2008 to 2010, are Japanese. It was a healthy control. All confirmatory study participants were diagnosed by a comprehensive ophthalmologic examination (autorefractor ARK-530A (Nidek), biometer / pachymeter AL-2000, UD-6000 (Tome), IOL Master (Carl Zeiss Meditec). use).

The research method adhered to the doctrine of the Declaration of Helsinki and was approved by the ethics committee of each participating institution. Details of the study were explained to all patients and healthy controls, and written informed consent was obtained from all participants.

Genotyping in the GWAS discovery stage GWAS genotyping for Japanese discovery samples is performed using the GeneChip Human Mapping 500K Array Set (500,568 SNPs) (Affymetrix) or Human OmniExpress chip (727,413 SNPs) (Illumina). It was based on the standard protocol recommended by the manufacturer. First, genotyping of 1,042 samples (504 patients with severe myopia and 538 healthy subjects) was performed using the GeneChip Human Mapping 500K Array Set. The remaining 2,227 samples (1,164 patients with severe myopia and 1,063 healthy subjects) were genotyped using the Human OmniExpress chip. Sample evaluation criteria were set to a minimum SNP call rate of 97% or higher, and samples with an SNP call rate of less than 97% were excluded. Regarding the SNP evaluation criteria, SNPs were excluded based on the following four quality control criteria.
・ Call rate less than 98% ・ Significant difference in missing data rate between patients with severe myopia and healthy subjects (P <1.0 × 10 ^-6 )
・ Frequency of minor allele is less than 1% ・ There is a large deviation from Hardy-Weinberg equilibrium (HWE) in the healthy group (P <0.0001)

In addition, potential relatives between samples were calculated based on homogeneity and excluded closely related samples with a PI-HAT value> 0.1875. 320,897 autosomal SNPs on the Affymetrix GeneChip Human Mapping 500K Array Set (494 patients with severe myopia, 538 healthy subjects) and 556,905 autosomal SNPs on the Illumina Human Omni Express chip that finally passed the above criteria. Imputation analysis was performed using (1,138 patients with severe myopia and 1,048 healthy subjects).

Genotype imputation
Imputation analysis of GWAS data was performed using Michigan Imputation Server (https://imputationserver.sph.umich.edu) [6]. 1000 Genomes Phase 3 v5 data (http://www.1000genomes.org) [7] was used as the reference panel for the imputation. Criteria for imputed SNPs are HWE P> 0.001, minor allele frequency> 1%, R-squared (coefficient of determination showing concordance between imputed genotype and actual genotype)> 0.7 Finally, 5,046,652 SNPs on the autosomal chromosome in 1,632 patients with severe myopia and 1,586 healthy subjects cleared the evaluation criteria and were used for statistical analysis.

Confirmation test
A confirmatory test was conducted to evaluate the outcome of the GWAS discovery stage. Based on the data from the previously conducted GWAS study [8,9] on the Japanese population, a confirmation test was conducted in the Japanese population.

Statistical analysis All relevant analyzes were performed using HelixTree SVS software (Golden Helix, Inc.). In order to avoid false positive results due to population stratification in the Japanese population of GWAS, the P value obtained in the GWAS stage was corrected by the inflation factor value (λ = 1.06). A meta-analysis between the GWAS and replication stage populations was performed using PLINK [10] under a fixed effects model. The threshold for genome-wide significance was set to P <5.0 × 10 ^-8 .

result
In the GWAS discovery stage, a total of 5,046,652 SNPs on autosomal chromosomes were associated with 1,632 Japanese patients with severe myopia and 1,586 Japanese healthy controls (Japanese population 1). As a result, in an allyl-based association study, 62 candidate gene regions that correlated with intensity myopia were identified at P <0.0001.

In order to verify the 62 candidate gene regions showing P <0.0001 identified in the GWAS stage, we used a Japanese population 2 (500 severe myopia patients, 4,869 healthy subjects) different from the above Japanese population 1. A confirmation test was conducted. Of the 62 candidate gene regions, SNPs in 9 candidate gene regions showed a genome-wide significance level (P <5.0 × 10 ^-8 ) in a meta-analysis of two Japanese populations. .. These 9 SNPs are shown in Table 2. The average number of risk SNPs possessed by 1,586 healthy subjects in the Japanese population 1 was calculated to be 4.39 ± 1.21. Of the nine candidate gene regions identified in this study, the ZC3H11B region of 1q41, the GJD2 region of 15q14, and the RASGRF1 region of 15q25.1 are the regions that correlate with the risk of developing myopia or myopia-related traits. Although it is a gene region that has been identified (Non-Patent Document 22-24), in this study, we newly identified an SNP that strongly correlates with intense myopia within these three gene regions.

References
1. Miyake M, Yamashiro K, Nakanishi H, Nakata I, Akagi-Kurashige Y, Kumagai K, et al. Evaluation of pigment epithelium-derived factor and complement factor I polymorphisms as a cause of choroidal neovascularization in highly myopic eyes. Vis Sci. 2013; 54 (6): 4208-4212.

2. Miyake M, Yamashiro K, Nakanishi H, Nakata I, Akagi-Kurashige Y, Tsujikawa A, et al. Association of paired box 6 with high myopia in Japanese. Mol Vis. 2012; 18: 2726-2735.

3. Miyake M, Yamashiro K, Nakanishi H, Nakata I, Akagi-Kurashige Y, Tsujikawa A, et al. Insulin-like growth factor 1 is not associated with high myopia in a large Japanese cohort. Mol Vis. 2013; 19: 1074-1081.

4. Miyake M, Yamashiro K, Tabara Y, Suda K, Morooka S, Nakanishi H, et al. Identification of myopia-associated WNT7B polymorphisms provides insights into the mechanism underlying the development of myopia. Nat Commun. 2015; 6: 6689.

5. Nakata I, Yamashiro K, Kawaguchi T, Nakanishi H, Akagi-Kurashige Y, Miyake M, et al. Calcium, ARMS2 genotype, and Chlamydia pneumoniae infection in early age-related macular degeneration: a multivariate analysis from the Nagahama study. Sci Rep. 2015; 5: 9345.

6. Das S, Forer L, Schonherr S, Sidore C, Locke AE, Kwong A, et al. Next-generation genotype imputation service and methods. Nat Genet. 2016; 48 (10): 1284-1287.

7. 1000 Genomes Project Consortium, Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, et al. An integrated map of genetic variation from 1,092 human genomes. Nature. 2012; 491 (7422): 56-65.

8. Khor CC, Miyake M, Chen LJ, Shi Y, Barathi VA, Qiao F, et al. Genome-wide association study identifies ZFHX1B as a susceptibility locus for severe myopia. Hum Mol Genet. 2013; 22 (25): 5288 -5294.

9. Hosoda Y, Yoshikawa M, Miyake M, Tabara Y, Shimada N, Zhao W, et al. CCDC102B confers risk of low vision and blindness in high myopia. Nat Commun. 2018; 9 (1): 1782.

10. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. 2007; 81 (3): 559-575.

Claims (3)

Using genomic DNA samples isolated from human subjects, the following single nucleotide polymorphisms (SNPs) in (1) to (9):
(1) SNP of SNP ID number rs72748160 at position 85,692,651 (position 201 in SEQ ID NO: 1) of chromosome 15.
(2) SNP of SNP ID number rs74633073 at position 69,229,747 of chromosome 3 (position 201 in SEQ ID NO: 2)
(3) SNP of SNP ID number rs76903431 at position 130,041,322 (position 201 in SEQ ID NO: 3) of chromosome 12.
(4) SNP of SNP ID number rs2246661 at position 204,974,863 (position 201 in SEQ ID NO: 4) of chromosome 1.
(5) SNP of SNP ID number rs12032649 at position 219,605,617 of chromosome 1 (position 201 in SEQ ID NO: 5)
(6) SNP of SNP ID number rs698047 at position 41,867,932 of chromosome 1 (position 201 in SEQ ID NO: 6)
(7) SNP of SNP ID number rs17029206 at position 1,739,832 of chromosome 3 (position 201 in SEQ ID NO: 7)
(8) SNP of SNP ID number rs589135 at position 34,709,241 (position 201 in SEQ ID NO: 8) of chromosome 15.
(9) SNP of SNP ID number rs28415942 at position 79,092,508 of chromosome 15 (position 201 in SEQ ID NO: 9)
A method for determining the risk of developing myopia, which involves examining the genotypes of at least 5 SNPs selected from the above, and the risk allele that is an indicator of the high risk of developing myopia is (1) T. (2) is T, (3) is A, (4) is C, (5) is G, (6) is G, (7) is T, (8) ) Is G and (9) is T, the method. The method according to claim 1 , comprising examining the genotypes of at least 6 SNPs among the above (1) to (9). The method according to claim 1 , which comprises examining the genotypes of the SNPs (1) to (9) above.