TWI807159B - Method for determination of risk of developing myopia - Google Patents

Abstract

The present inventors intensively conducted genetic analysis with Japanese people, who have a high prevalence of high myopia, using samples obtained from an enormous number of high myopia patients and healthy individuals, to successfully identify nine single nucleotide polymorphisms (SNPs) correlated with high myopia at a genome-wide significance level. SNPs showing a significant correlation with a risk of developing high myopia are considered to be SNPs that have a strong genetic effect on the risk of developing high myopia. Using such SNPs as indicators, a genetic influence on the risk of developing high myopia can be assessed more appropriately.

Description

Methods for determining the risk of developing myopia

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

Myopia is defined as those whose spherical equivalent (SE) is -0.5D (diopters) or more, and is the most common refractive error in the world. High myopia is generally defined as SE<-6.0D or axial length>26.0mm, which increases the risk of various eye diseases and systemic diseases such as retinal detachment, glaucoma, cataract, macular hemorrhage, macular degeneration, etc. that may cause blindness or severe visual impairment (non-patent literature 1, 2). It has been reported that myopia is more common in Asians than non-Asians (Non-Patent Documents 3-8) and compared with non-Asian countries (2.0-2.3%), the prevalence of high myopia among young adults is extremely high in Asia (6.8-21.6%) (Non-Patent Document 9). In the past few decades, the prevalence of myopia has continued to rise rapidly all over the world (Non-Patent Document 10). According to recent reports, it is predicted that by 2050, the number of myopia patients in the world should reach about 5 billion (about half of the world's population), and the number of high myopia patients should reach about 1 billion (about 10% of the world's population) (Non-Patent Document 11). Although the etiology of myopia has not been precisely elucidated, it is considered that myopia is a multifactorial disease in which various environmental factors and genetic factors participate in the onset (Non-Patent Documents 12-17). It is implied that the higher the degree of myopia, the stronger the influence of genetic factors is (non-patent literature 18-20).

Genome-wide association study (GWAS) is aimed at genotypes covering all regions of the genome, and searching for genotypes that show significant frequency differences between unrelated patient groups and healthy groups is very effective in elucidating the genetic factors of multifactorial diseases. Since 2009, multiple GWASs targeting myopia have been implemented, and so far, many susceptibility gene regions for myopia and myopia-related characteristics have been identified in various ethnic groups (non-patent literature 21-24). In the NHGRI-EBI GWAS Catalog (https://www.ebi.ac.uk/gwas/), a database that collects the results of GWAS studies reported as papers, about 200 candidate gene regions related to myopia and myopia-related characteristics have been reported (Non-Patent Document 25). However, it is estimated that these approximately 200 candidate gene regions are only a part of the repertoire of genetic factors for myopia and myopia-related traits. According to two papers in 2013 related to the large-scale GWAS of myopia and refractive error, more than 30 susceptibility gene regions have been reported (Non-patent literature 26, 27), but it is estimated that these gene regions are not even as good as 12% of the total phenotype dispersion of myopia and refractive error (Non-patent literature 17). Furthermore, it is considered that in most of the approximately 200 candidate gene regions, the strength of the correlation between myopia or myopia-related features and the candidate gene region, or the presence or absence of the correlation is not the same between ethnic groups or the severity of myopia. These facts imply that there are many unidentified genetic factors that affect myopia and myopia-related characteristics. [Prior Art Literature] [Non-patent literature]

[Non-Patent Document 1] Saw SM, Gazzard G, Shih-Yen EC, Chua WH. Myopia and associated pathological complications. Ophthalmic Physiol Opt. 2005;25(5):381-391. [Non-Patent Document 2] Burton TC. The influence of refractive error and lattice degeneration on the incidence of retinal detachment. Trans Am Ophthalmol Soc. 1989;87:143-157. [Non-Patent Document 3] Wilson A, Woo G. A review of the prevalence and causes of myopia. Singapore Med J. 1989;30(5):479-484. [Non-Patent Document 4]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. [Non-Patent Document 5] 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. [Non-Patent Document 6] 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. [Non-Patent Document 7] Pan CW, Klein BE, Cotch MF, Shrager S, Klein R, Folsom A, et al. Racial variations in the prevalence of reactive errors in the United States: the multi-ethnic study of atherosclerosis. Am J Ophthalmol. 2013;155(6):1129 -1138. [Non-Patent Document 8] 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):25 90-2598. [Non-Patent Document 9] Wong YL, Saw SM. Epidemiology of Pathologic Myopia in Asia and Worldwide. Asia Pac J Ophthalmol (Phila). 2016;5(6):394-402. [Non-Patent Document 10] Dolgin E. The myopia boom. Nature. 2015;519(7543):276-278. [Non-Patent Document 11] 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):1036-10 42. [Non-Patent Document 12] Baird PN, Schache M, Dirani M. The GEnes in Myopia (GEM) study in understanding the aetiology of reactive errors. Prog Retin Eye Res. 2010;29(6):520-542. [Non-Patent Document 13] Au Eong KG, Tay TH, Lim MK. Education and myopia in 110,236 young Singaporean males. Singapore Med J. 1993;34(6):489-492. [Non-Patent Document 14] 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. [Non-Patent Document 15] Morgan IG, Rose KA. Myopia and international educational performance. Ophthalmic Physiolog Opt. 2013;33(3):329-338. [Non-Patent Document 16] French AN, Ashby RS, Morgan IG, Rose KA. Time outdoors and the prevention of myopia. Exp Eye Res. 2013;114:56-68. [Non-Patent Document 17] 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 Comm un. 2016;7:11008. [Non-Patent Document 18] 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. [Non-Patent Document 19] 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. [Non-Patent Document 20] Peet JA, Cotch MF, Wojciechowski R, Bailey-Wilson JE, Stambolian D. Heritability and familial aggregation of reactive error in the Old Order Amish. Invest Ophthalmol Vis Sci. 2007;48(9):4002-4006. [Non-Patent Document 21] Li J, Zhang Q. Insight into the molecular genetics of myopia. Mol Vis. 2017;23:1048-1080. [Non-Patent Document 22] 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. [Non-Patent Document 23] 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 reactive errors and myopia at 15q14. Nat Genet. 2010 ;42(10):897-901. [Non-Patent Document 24]Hysi PG, Young TL, Mackey DA, Andrew T, Fernandez-Medarde A, Solouki AM, et al. A genome-wide association study for myopia and reactive error identifies a susceptibility locus at 15q25. Nat Genet. 2010 ;42(10):902-905. [Non-Patent Document 25]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. [Non-Patent Document 26]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. [Non-Patent Document 27] 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.

[Problem to be solved by the invention]

As mentioned above, myopia is considered to be a disease caused by the complex participation of genetic factors and environmental factors (Non-Patent Documents 12-17), and it is suggested that the influence of genetic factors is stronger as the intensity of myopia is higher (Non-Patent Documents 18-20). The genotypes that have been reported to be associated with the risk of developing myopia are genotypes that have been identified through genetic research on a myopia case group that mainly includes low or moderate myopia, but the low to moderate myopia case group includes many cases that are greatly influenced by environmental factors. The genotypes identified for this case group are not necessarily effective as genotypes for determining the risk of myopia since it is difficult to say that the polytype has a high genetic effect on the onset of myopia. Also, with such genotype polytypes, it is difficult to accurately determine the risk of myopia in areas where the prevalence of high myopia is high. The purpose of the present invention is to provide a means for more accurately determining the onset risk of myopia in various regions and races including regions with a high prevalence of high myopia than in the prior art. [Means to solve the problem]

The inventors of this case took the Japanese with a high prevalence of high myopia as objects, obtained samples from a large number of high myopia patients and healthy people, and performed gene analysis results exclusively, so that they identified 9 single nucleotide polymorphisms (SNPs) that were related to high myopia at a significant level in the whole genome, and completed the invention of this case.

That is, the present invention provides a method for determining the risk of myopia, which comprises using a genome DNA sample isolated from a human subject, and investigating the genotype of at least one SNP selected from the following (1) to (9): (1) The SNP with SNP ID number rs72748160 located at position 85,692,651 of chromosome 15 (position 201 in Sequence ID No. 1), or a SNP with linkage disequilibrium (linkage disequilibrium) with it, (2) SNP ID number rs74633073 located at position 69,229,747 of chromosome 3 (position 201 in Sequence ID No. 2), or a SNP in linkage disequilibrium with it, (3) SNP ID number rs76903431 located at position 130,041,322 of chromosome 12 (position 201 in Sequence ID No. 3), or a SNP in linkage disequilibrium with it, (4) SNP with SNP ID number rs2246661 located at position 204,974,863 of chromosome 1 (position 201 in Sequence ID No. 4), or a SNP in linkage disequilibrium with it, (5) SNP with SNP ID number rs12032649 located at position 219,605,617 of chromosome 1 (position 201 in Sequence ID No. 5), or a SNP in linkage disequilibrium with it, (6) SNP with SNP ID number rs698047 located at position 41,867,932 of chromosome 1 (position 201 in Sequence ID number 6), or a SNP in linkage disequilibrium with it, (7) SNP with SNP ID number rs17029206 located at position 1,739,832 of chromosome 3 (position 201 in Sequence ID No. 7), or a SNP in linkage disequilibrium with it, (8) SNP with SNP ID number rs589135 located at position 34,709,241 of chromosome 15 (position 201 in Sequence ID No. 8), or a SNP in linkage disequilibrium with it, (9) SNP with SNP ID number rs28415942 located at position 79,092,508 of chromosome 15 (position 201 in Sequence ID No. 9), or a SNP in linkage disequilibrium with it, (Except for the method of investigating only the genotypes of 1 to 3 SNPs selected from the SNPs in linkage disequilibrium with the SNP of (5), the SNPs in linkage disequilibrium with the SNP of (8), and the SNPs in linkage disequilibrium with the SNP of (9).) In addition, the present invention provides a method for determining the risk of myopia, which includes using a genome DNA sample isolated from a human subject, and investigating the genotype of at least one SNP selected from the following (1) to (9): (1) SNP located at position 85,692,651 of chromosome 15 (position 201 in Sequence ID No. 1) with SNP ID number rs72748160, (2) The SNP with SNP ID number rs74633073 located at position 69,229,747 of chromosome 3 (position 201 in Sequence ID No. 2), (3) The SNP of SNP ID number rs76903431 located at position 130,041,322 of chromosome 12 (position 201 in sequence identification number 3), (4) The SNP of SNP ID number rs2246661 located at position 204,974,863 of chromosome 1 (position 201 in Sequence ID No. 4), (5) The SNP of SNP ID number rs12032649 located at position 219,605,617 of chromosome 1 (position 201 in Sequence ID No. 5), (6) The SNP of SNP ID number rs698047 located at position 41,867,932 of chromosome 1 (position 201 in Sequence ID number 6), (7) The SNP of SNP ID number rs17029206 located at position 1,739,832 of chromosome 3 (position 201 in Sequence ID No. 7), (8) The SNP of SNP ID number rs589135 located at position 34,709,241 of chromosome 15 (position 201 in Sequence ID No. 8), (9) The SNP of SNP ID number rs28415942 located at position 79,092,508 of chromosome 15 (position 201 in Sequence ID No. 9), The risk allele (risk allele) line that becomes a high-risk indicator of onset of myopia: (1) is T, (2) is T, (3) is A, (4) is C, (5) is G, (6) is G, (7) is T, (8) is G, (9) is T. [Effect of Invention]

According to the present invention, it is possible to provide a means for determining the onset risk of myopia more accurately than the prior art. The SNPs that are significantly associated with the risk of developing myopia are SNPs that have a high genetic effect on the risk of developing myopia. By using this SNP as an indicator, the influence of genetics on the risk of myopia can be more appropriately evaluated, and the risk of myopia can be determined more accurately than in the prior art (assisting the risk determination of myopia by doctors and the like). In addition to the genetic examination for the purpose of understanding the risk of myopia itself, the present invention can be implemented for subjects before the onset of myopia, and it is helpful for the early implementation of the following: life guidance for suppressing myopia (suggestions for improving the living environment such as computers, smart phones, TV games, reading, etc.), or medical treatment for suppressing myopia such as orthokeratology and low-concentration atropine treatment.

[Mode for Carrying Out the Invention]

Table 1 shows the nine SNPs identified by the inventors of the present case that are significantly associated with high myopia.

[Table 1] SNPs chromosomal region Chromosomal location (GRCh38) gene region allele risk allele P value Odds ratio ^* Sequence listing ^** (1) rs72748160 15q25.3 85,692,651 AKAP13 G/T T 3.76E-10 1.93 Serial ID 1 (2) rs74633073 3p14.1 69,229,747 FRMD4B C/T T 6.43E-13 1.91 SIN2 (3) rs76903431 12q24.33 130,041,322 LINC02418 A/T A 1.41E-09 1.66 SIN3 (4) rs2246661 1q32.1 204,974,863 NFASC C/T C 2.48E-09 1.31 SIN4 (5) rs12032649 1q41 219,605,617 ZC3H11B G/T G 1.58E-11 1.31 SIN 5 (6) rs698047 1p34.2 41,867,932 HIVEP3 C/G G 4.35E-10 1.28 SIN6 (7) rs17029206 3p26.3 1,739,832 CNTN4-CNTN6 C/T T 2.25E-09 1.28 SIN 7 (8) rs589135 15q14 34,709,241 GJD2 A/G G 4.16E-10 1.28 SIN 8 (9) rs28415942 15q25.1 79,092,508 RASGRF1 C/T T 3.19E-09 1.27 Serial Identification Number 9 ^* In the example, odds ratios of risk alleles calculated by meta-analysis targeting Japanese groups 1 and 2. ^** The position of any SNP is 201.

In addition, in this specification, the position on the chromosome of an arbitrary base is shown based on the human genome reference sequence GRCh38. The SNP ID number starting with rs is the reference SNP ID number in dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/) of the SNP database of the National Center for Biotechnology Information (NCBI). Those with ordinary knowledge in the technical field can easily obtain information such as the physical position on the chromosome of the SNP and its nearby base sequence based on the SNP ID number. The sequence identification numbers 1 to 9 in the sequence listing of this case show the 200 bp base sequence of the genome before and after each SNP, but the base sequence of the genome adjacent to these regions can be easily obtained by those with ordinary knowledge in the technical field.

The SNPs shown in Table 1 were comprehensively identified as SNPs significantly associated with high myopia by GWAS targeting the high myopia patient group and the healthy group of Japanese. However, the reason for targeting the Japanese group is that the prevalence rate of high myopia in the Asian region including Japan shows a significantly higher value than other regions, and many samples of high myopia patients who are considered to be particularly affected by genetic factors can be obtained. The people who can correctly determine the risk of myopia by the method of the present invention are not limited to Japanese, and it is also possible to determine people in Asian regions other than Japan, and people in other non-Asian regions in Europe and the United States. Therefore, the subjects who can implement the method of the present invention broadly include Asians including Japanese, and non-Asians such as Europeans and Americans. When the method of the present invention is applied to the suppression of myopia of subjects, young subjects (for example, under the age of 15) can be the main subjects.

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 or more, or all nine genotypes are investigated. Since the average number of retained risk alleles calculated from 1,586 healthy subjects is 4.39±1.21 (refer to the following example), it is expected that at least 5 genotypes, preferably 9 genotypes, can be investigated. In the case where only a part of the 9 samples are investigated, any SNP can be selected, but the one with the highest odds ratio, that is, the SNP listed in the upper rank in Table 1, can be selected preferentially. For example, when examining the genotypes of five or more SNPs among (1) to (9), it is preferable to select five or more SNPs including (1) to (5).

In addition, instead of the SNPs of (1) to (9), the genotype of the SNP in linkage disequilibrium with each SNP may be investigated. As is well known in the art, when a plurality of SNPs are in strong linkage disequilibrium, those SNPs show the same behavior. For example, when SNP-A, SNP-B, and SNP-C are in a strong linkage disequilibrium state, when SNP-A shows a significant correlation with disease X, SNP-B and SNP-C also show a significant correlation with disease X. Therefore, if it is a SNP in a strong linkage disequilibrium state with each of the SNPs (1) to (9), it can be used to determine the risk of myopia because it is significantly associated with high myopia similarly to the SNPs of (1) to (9).

As a value indicating the strength of linkage disequilibrium, linkage disequilibrium coefficients such as D' and r ² are known. D' and ^r2 can be obtained by the calculation method of 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.). In general, SNPs with a relationship of r ² >0.7 are considered to be in a state of strong linkage disequilibrium. In the present invention, "a SNP in linkage disequilibrium" with a certain SNP refers to a SNP having a relationship of r ² >0.7 with the SNP. Information on specific SNPs and "SNPs that are in linkage disequilibrium (SNPs that are in the relationship of r ² >0.7)" can be easily searched in, for example, LDproxy (https://ldlink.nci.nih.gov/?tab=ldproxy) in LDlink (https://ldlink.nci.nih.gov/) of the National Cancer Institute (NCI)'s linkage disequilibrium database.

In the present invention, when investigating the genotypes of multiple SNPs, for example, the number of genotypes (high-risk genotypes) with risk alleles (alleles showing a positive correlation with the onset risk of high myopia) can be used to determine the myopia risk of the subject. In this case, the more the number of high-risk genotypes, the higher the risk of myopia. Since the average number of high-risk genotypes in healthy people is 4.39±1.21, "5 or more high-risk genotypes" exceeding this average value can be used as a criterion for judging that the risk of myopia is high.

Furthermore, each risk allele can be weighted according to odds ratios. Even among the subjects with 2 high-risk genotypes, if the subjects A with 2 high-risk genotypes for (4) (rs2246661, odds ratio 1.31) and (6) (rs698047, odds ratio 1.28) were compared with those for (1) (rs72748160, odds ratio 1.93) and (2) (rs74633073, odds ratio 1.91) If there are two subjects B with high-risk genotypes, it can be evaluated that subject B has a higher risk of developing myopia. In the method of the present invention, in addition to the number of high-risk genotypes, the height of the odds ratio of the detected high-risk genotypes (the type and combination of high-risk genotypes) can also be considered to determine the risk of myopia. For example, the following judgment method can be used: the odds ratio, or an appropriate value corresponding to the odds ratio, is set as the score of the risk allele of each SNP, and the scores of the high-risk genotypes detected by the subject are added or accumulated, and the risk of myopia of the subject is evaluated by score. The odds ratios shown in Table 1 can be used, and many high myopia patients can be further analyzed to calculate the odds ratios, and the obtained values can be used instead of the odds ratios in Table 1.

Genomic DNA samples used in the method of the present invention can be easily prepared from peripheral blood or oral mucosa, etc., collected from subjects by conventional methods.

The method for investigating the genotype of a SNP is known per se, and the method is not particularly limited in the present invention. For example, the genotype can be determined by sequencing the nucleotide sequence of the SNP site. Also, various methods such as invasion assay, TaqMan-PCR method, Allele Specific Primer (ASP) method, and a wafer carrying a probe for SNP detection are known as SNP typing methods. Either of these methods is a common method, and various kits containing reagents necessary for these methods are also commercially available. When investigating the genotype of a SNP in the present invention, any known method can be used. Primers, probes, etc. necessary for genotype detection can be easily designed and prepared based on the base sequence information near the SNP by those with ordinary knowledge in this technical field.

The probe for SNP detection used in the SNP typing method of the TAQMAN-PCR method, etc., usually has an additional nucleic acid part of the additional or adapter or adapter, which is connected to the structure that is connected to the structure of the base sequence of alkali base (hybridization part of the target region). The alkali base sequence of several alkali -based genes of the SNP of the object of the object is the same base sequence of the alkali base sequence of parts of the gene body, or the alkali -based sequence that is complementary to it. The full length of the part that hybridizes to the target gene body region (the full length of the probe when the additional nucleic acid part is not included) is about 15 to 30 bases, for example, about 16 to 30 bases, about 17 to 30 bases, or about 18 to 30 bases.

(1) The probe used to detect the genotype of rs72748160 contains a nucleic acid part composed of the following base sequence, as a part that hybridizes with the target gene body region. For example, it may be a probe composed of a base sequence identical to 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 base sequence complementary thereto. The probe in this case includes TTAG[G]GCAA, TTAG[T]GCAA, TTGC[C]CTAA, or TTGC[A]CTAA as the sequence at positions 197-205 in SEQ ID NO: 1 or a sequence complementary thereto ([ ] is a SNP part).

(2) The probe used to detect the genotype of rs74633073 includes a nucleic acid part composed of the following base sequence, as a part that hybridizes with the target gene body region. The base sequence is a base sequence of about 15 to 30 bases including the 201st position of SEQ ID NO: 2 (the SNP of rs74633073) and the number of bases before and after it, or a base sequence complementary to it. For example, it may be a probe composed of a base sequence identical to 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 base sequence complementary thereto. The probe in this case includes CTGT[C]GCCA, CTGT[T]GCCA, TGGC[G]ACAG, or TGGC[A]ACAG ([ ] is a SNP part) as the sequence at positions 197 to 205 in SEQ ID NO: 2 or a sequence complementary thereto.

(3) The probe used to detect the genotype of rs76903431 includes a nucleic acid portion composed of the following base sequence, as a portion that hybridizes with the target gene body region. The base sequence includes a base sequence of about 15 to 30 bases in sequence identification number 3 (the SNP of rs76903431) and the number of bases before and after it, or a base sequence complementary to it. For example, it may be a probe composed of a base sequence identical to 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 base sequence complementary thereto. The probe in this case includes ATAG[A]TCAA, ATAG[T]TCAA, TTGA[T]CTAT, or TTGA[A]CTAT ([ ] is a SNP part) as the sequence at positions 197 to 205 in SEQ ID NO: 3 or a sequence complementary thereto.

(4) The probe used to detect the genotype of rs2246661 includes a nucleic acid part composed of the following base sequence, as a part that hybridizes with the target gene body region, and the base sequence includes a base sequence of about 15 to 30 bases including the 201st position of SEQ ID NO: 4 (the SNP of rs2246661) and the number of bases before and after, or a base sequence complementary to it. For example, it may be a probe composed of a base sequence identical to 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 base sequence complementary thereto. In this case, the probe includes GGGA[C]AAGG, GGGA[T]AAGG, CCTT[G]TCCC, or CCTT[A]TCCC ([ ] is a SNP part) as the sequence at positions 197 to 205 in SEQ ID NO: 4 or a sequence complementary thereto.

(5) The probe used to detect the genotype of rs12032649 includes a nucleic acid part composed of the following base sequence, as a part that hybridizes with the target gene body region. The base sequence is a base sequence of about 15 to 30 bases including the 201st position of sequence identification number 5 (the SNP of rs12032649) and the number of bases before and after, or a base sequence complementary to it. For example, it may be a probe composed of a base sequence identical to 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 base sequence complementary thereto. The probe in this case includes GCCA[G]CTGG, GCCA[T]CTGG, CCAG[C]TGGC, or CCAG[A]TGGC ([ ] is a SNP part) as the sequence at positions 197 to 205 in SEQ ID NO: 5 or a sequence complementary thereto.

(6) The probe used to detect the genotype of rs698047 includes a nucleic acid part composed of the following base sequence, as a part that hybridizes with the target gene body region. The base sequence includes a base sequence of about 15 to 30 bases including the 201st position of SEQ ID NO. For example, it may be a probe composed of a base sequence identical to 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 base sequence complementary thereto. In this case, the probe includes CCCC[C]TCCC, CCCC[G]TCCC, GGGA[G]GGGG, or GGGA[C]GGGG ([ ] is a SNP part) as the sequence at positions 197 to 205 in SEQ ID NO: 6 or a sequence complementary thereto.

(7) The probe used to detect the genotype of rs17029206 includes a nucleic acid part composed of the following base sequence, as a part that hybridizes with the target gene body region. The base sequence includes a base sequence of about 15 to 30 bases including the 201st position of sequence identification number 7 (the SNP of rs17029206) and the preceding and following bases, or a base sequence complementary to it. For example, it may be a probe composed of a base sequence identical to 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 base sequence complementary thereto. The probe in this case includes GAAA[C]TTAC, GAAA[T]TTAC, GTAA[G]TTTC, or GTAA[A]TTTC ([ ] is a SNP part) as the sequence at positions 197-205 in SEQ ID NO: 7 or a sequence complementary thereto.

(8) The probe used to detect the genotype of rs589135 contains a nucleic acid part composed of the following base sequence, as a part that hybridizes with the target gene body region, and the base sequence includes a base sequence of about 15 to 30 bases including the 201st position of sequence identification number 8 (the SNP of rs589135) and the number of bases before and after, or a base sequence complementary to it. For example, it may be a probe composed of a base sequence identical to 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 base sequence complementary thereto. The probe in this case includes GAAG[A]GGCT, GAAG[G]GGCT, AGCC[T]CTTC, or AGCC[C]CTTC ([ ] is a SNP part) as the sequence at positions 197-205 in SEQ ID NO: 8 or a sequence complementary thereto.

(9) The probe used to detect the genotype of rs28415942 includes a nucleic acid part composed of the following base sequence, as a part that hybridizes with the target gene body region. The base sequence is a base sequence of about 15 to 30 bases including the 201st position of sequence identification number 9 (the SNP of rs28415942) and the number of bases before and after, or a base sequence complementary to it. For example, it may be a probe composed of a base sequence identical to 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 base sequence complementary thereto. The probe in this case is the sequence at positions 197 to 205 in SEQ ID NO: 9 or a sequence complementary thereto, and includes CACA[C]CCAC, CACA[T]CCAC, GTGG[G]TGTG, or GTGG[A]TGTG ([ ] is a SNP part).

The method for determining the onset risk of high myopia according to the present invention may be implemented in combination with other genetic diagnosis methods. For example, using a microarray chip combining a probe for detecting genotypes of polytypes associated with risk of disease or constitution, and a probe for detecting SNPs shown in Table 1, the determination of the onset risk of high myopia according to the present invention can be performed as one of various items of genetic testing.

According to the present invention, information or advice that the risk of myopia is high can be provided to subjects judged to be at high risk. By providing this information, it is possible to implement early myopia prevention advice by improving the living environment (computer, smart phone, TV game, reading environment, etc.), or myopia suppression by orthokeratology or low-concentration atropine treatment, etc. for the prevention or suppression of myopia. [Example]

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

Materials and methods object A total of 1,668 unrelated Japanese patients with high myopia (SE > -9.0D in at least one eye) and a total of 1,601 unrelated Japanese healthy controls (both eyes -0.5D > SE > +2.0D) were included in the discovery stage of GWAS. Cases of patients and healthy subjects were collected at Yokohama City University, Okada Ophthalmology Department and Aoto Ophthalmology Department in Yokohama City, Kanagawa Prefecture. All subjects were diagnosed by comprehensive ophthalmic examination (ARK-730A (NIDEK), ARK-700A (NIDEK), KP-8100P (TOPCON) for autorefractor and AL-2000 (Tomey) for biometer/pachymeter) including axial length (AL), fundus examination, spherical refractive power, and corneal curvature. Patients with high myopia have been identified as having no known hereditary disease (glaucoma, keratoconus, Marfan's syndrome, etc.) associated with myopia or at least one of high myopia. The QIAamp DNA Blood Maxi Kit (QIAGEN) was used for the collection of peripheral blood lymphocytes and the extraction of genomic DNA from peripheral blood cells. In order to prevent the fluctuation of DNA quality, the experimental operation was carried out under standard conditions.

For the replication stage (validation test), an unrelated Japanese group (500 patients with AL > 26.0 mm in both eyes and 4,869 healthy controls) different from the GWAS stage was added. Japanese high myopia cases were collected from the Kyoto high myopia cohort (cohort) [1-3]. In addition, it will be recruited from general citizens living in Nagahama City, Shiga Prefecture from 2008 to 2010. Generally healthy subjects of the Nagahama Study (Human bioscience) data set[4,5] were set as Japanese healthy control group. The verification test participants were all diagnosed by comprehensive ophthalmic examination (ARK-530A (NIDEK) for autorefractor, AL-2000, UD-6000 (Tomey), IOL Master (Carl Zeiss Meditec) for biometer/pachymeter ).

The research methods followed the teachings of the Helsinki Declaration and were approved by the ethics committees of participating facilities. The details of the study were explained to all the patients and healthy subjects in the control group, and written informed consent was obtained from all the participants.

Genotyping in the Exploration Phase of GWAS GWAS genotyping for Japanese exploratory samples was performed using GeneChip Human Mapping 500K Array Set (500,568 SNPs) (Affymetrix) or Human OmniExpress chip (727,413 SNPs) (Illumina) according to the recommended standard procedure of each manufacturer. First, using the GeneChip Human Mapping 500K Array Set, 1,042 samples (504 high myopia patients and 538 healthy subjects) were genotyped. Human OmniExpress chip was used for genotyping of the remaining 2,227 samples (1,164 high myopia patients and 1,063 healthy subjects). The evaluation standard of the samples was set as the minimum SNP call rate (call rate) above 97%, and the samples with the SNP call rate lower than 97% were excluded. In addition, regarding the evaluation criteria of SNPs, SNPs were excluded based on the following four quality control criteria.・The interpretation rate is lower than 98% ・There is a significant difference in the missing data rate between high myopia patients and healthy people (P > 1.0×10 ^-6 ) ・The frequency of the minor allele (minor allele frequency) is less than 1%

Again, potential relatedness between samples was calculated based on homology, and samples showing relatedness with a PI-HAT value > 0.1875 were excluded. Finally, 320,897 autosome SNPs on the Affymetrix GeneChip Human Mapping 500K Array Set (494 high myopia patients, 538 healthy subjects) and 556,905 autosome SNPs (1,138 high myopia patients, 1,048 healthy subjects) on the Illumina Human OmniExpress chip that passed the above evaluation criteria were carried out Imputation analysis.

Genotype (genotype) interpolation Complementary analysis of GWAS data was performed using Michigan Imputation Server (https://imputationserver.sph.umich.edu) [6]. For the reference panel (Reference panel) of interpolation, the data of 1000 Genomes Phase 3 v5 (http://www.1000genomes.org) [7] was used. Set the evaluation criteria of the interpolated SNP as HWE P > 0.001, secondary allele frequency > 1%, R-squared (displays the coefficient of determination of the concordance rate between the interpolated genotype and the actual genotype) > 0.7. Finally, 5,046,652 SNPs on somatic chromosomes of 1,632 highly myopic patients and 1,586 healthy individuals passed the evaluation criteria and were used for statistical analysis.

Verification test Validation experiments were performed to evaluate the results of the exploratory phase of the GWAS. Based on the data of the previous GWAS studies [8,9] on the Japanese group, a verification test of the Japanese group was carried out.

Statistical Analysis All correlation analyzes were performed using HelixTree SVS software (Golden Helix, Inc.). In order to avoid the false positive result of GWAS caused by group stratification in the Japanese group, the P value obtained in the GWAS stage was corrected with the value of the inflation factor (λ= 1.06). The meta-analysis between groups in the GWAS phase and replicate phase was performed under a fixed-effects model using PLINK [10]. The threshold value of the genome-wide significance level was set at P > 5.0×10 ^-8 .

result In the exploration stage of GWAS, 1,632 Japanese patients with high myopia and 1,586 Japanese healthy controls (Japanese group 1) were used as objects, and a total of 5,046,652 SNPs on the somatic chromosome were analyzed. As a result, 62 candidate gene regions that were associated with high myopia at P > 0.0001 were identified in the correlation analysis of the allele bases.

In order to examine the 62 candidate gene regions that showed P > 0.0001 identified in the GWAS stage, a verification test was carried out using Japanese group 2 (500 highly myopic patients and 4,869 healthy individuals) different from the above-mentioned Japanese group 1. Among the 62 candidate gene regions, the SNPs in the 9 candidate gene regions showed a genome-wide significant level (P > 5.0×10 ^-8 ) correlation in the integrated analysis of 2 Japanese groups. These 9 SNPs are shown in Table 2. The result of calculating the average number of risk SNPs with 1,586 healthy individuals from the Japanese group 1 was 4.39±1.21. Among the 9 candidate gene regions identified in this study, the 3 gene regions of the ZC3H11B region of 1q41, the GJD2 region of 15q14, and the RASGRF1 region of 15q25.1 are gene regions identified as regions associated with the risk of myopia or myopia-related characteristics (non-patent literature 22-24).

[Table 2] In the Japanese group, the SNPs associated with high myopia were shown at the genome-wide significance level ( P > 5×10 ^-8 ) Consolidated Analysis Targeting Japanese Groups 1 and 2 Japanese group 1: 1,632 people with high myopia and 1,586 healthy people Japanese group 2: 500 people with high myopia and 4,869 healthy people risk allele frequency risk allele frequency SNPs chromosomal region Chromosomal location (GRCh38) gene region allele risk allele P value Odds patient healthy person P value Odds patient healthy person P value Odds rs698047 1p34.2 41,867,932 HIVEP3 C/G G 4.35E-10 1.28 0.512 0.451 8.13E-07 1.28 0.524 0.460 1.22E-04 1.29 rs2246661 1q32.1 204,974,863 NFASC C/T C 2.48E-09 1.31 0.278 0.233 3.25E-05 1.27 0.276 0.215 1.07E-05 1.39 rs12032649 1q41 219,605,617 ZC3H11B G/T G 1.58E-11 1.31 0.423 0.493 1.33E-08 1.33 0.418 0.479 2.43E-04 1.28 rs17029206 3p26.3 1,739,832 CNTN4-CNTN6 C/T T 2.25E-09 1.28 0.346 0.398 1.43E-05 1.25 0.330 0.398 2.56E-05 1.34 rs74633073 3p14.1 69,229,747 FRMD4B C/T T 6.43E-13 1.91 0.063 0.041 5.06E-05 1.59 0.064 0.026 1.98E-11 2.54 rs76903431 12q24.33 130,041,322 LINC02418 A/T A 1.41E-09 1.66 0.077 0.049 2.27E-06 1.64 0.068 0.042 1.06E-04 1.68 rs589135 15q14 34,709,241 GJD2 A/G G 4.16E-10 1.28 0.508 0.452 5.73E-06 1.25 0.490 0.418 1.17E-05 1.34 rs28415942 15q25.1 79,092,508 RASGRF1 C/T T 3.19E-09 1.27 0.422 0.483 1.09E-06 1.28 0.404 0.460 7.31E-04 1.26 rs72748160 15q25.3 85,692,651 AKAP13 G/T T 3.76E-10 1.93 0.051 0.028 1.92E-06 1.88 0.044 0.022 2.17E-05 2.02 Japanese group 1: group using genome-wide association analysis (GWAS) Japanese group 2: the group used in the replication study

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. Invest O phthalmol 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 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.

none.

none.

<110> SEED Co.,Ltd.

<120> Methods for determining myopia risk

<130> PF663-TW

<140> TW 109102744

<141> 2020-01-30

<150> JP 2019-015193

<151> 2019-01-31

<160> 9

<170> PatentIn Version 3.5

<210> 1

<211> 401

<212>DNA

<213> Homo sapiens

<220>

<221> Variation

<222> (201)..(201)

<223> k is G or T

<400> 1

<210> 2

<211> 401

<212>DNA

<213> Homo sapiens

<220>

<221> Variation

<222> (201)..(201)

<223> y is C or T

none.

Claims (3)

A method for determining the risk of onset of myopia, the method comprising using a genome DNA sample isolated from a human subject, and investigating the genotypes of at least 5 SNPs selected from the following (1) to (9) single nucleotide polymorphisms (SNPs), wherein the risk allele (risk allele) that becomes a high risk indicator of onset of myopia is (1) T, (2) T, (3) A, (4) C, (5) G, (6) G, (7) T , (8) is G, (9) is T: (1) the SNP of SNP ID number rs72748160 located at position 85,692,651 of chromosome 15 (position 201 in sequence identification number 1); (2) the SNP of SNP ID number rs74633073 located at position 69,229,747 of chromosome 3 (position 201 of sequence identification number 2); (3) the SNP located on chromosome 12 (4) SNP located at position 204,974,863 of chromosome 1 (position 201 in sequence identification number 4) with SNP ID number rs2246661; (5) located at position 219,605,617 of chromosome 1 (position 20 in sequence identification number 5) 1) the SNP with SNP ID number rs12032649; (6) the SNP with SNP ID number rs698047 located at position 41,867,932 of chromosome 1 (position 201 in sequence identification number 6); (7) the SNP with SNP ID number rs17029206 located at position 1,739,832 of chromosome 3 (position 201 in sequence identification number 7); The SNP of SNP ID number rs589135 at position 34,709,241 of chromosome 15 (position 201 in sequence identification number 8); (9) the SNP of SNP ID number rs28415942 at position 79,092,508 of chromosome 15 (position 201 in sequence identification number 9). The method according to claim 1, which comprises investigating the genotypes of at least 6 SNPs in the aforementioned (1) to (9). The method according to claim 1, comprising investigating the genotypes of the SNPs in (1) to (9).