JPWO2011148715A1 - Normal-tension glaucoma disease susceptibility gene and use thereof - Google Patents

Abstract

A normal-tension glaucoma disease susceptibility gene and a method for using the same Single nucleotide polymorphism international numbers rs3213787, rs735860 from among polymorphic sites that were found by comprehensive analysis and comparison of single nucleotide polymorphisms in the entire genome for many patients with normal tension glaucoma and normal individuals The polymorphic sites of rs4412249 and rs2763979 were found to be effective for diagnosis.

Description

  The present invention relates to a normal-tension glaucoma disease susceptibility gene and use thereof.

  Glaucoma is a progressive refractory disease that damages the optic nerve due to an increase in intraocular pressure that exceeds normal ocular pressure that can maintain normal visual function. If left unattended, this is a disease that can cause visual field stenosis, which can cause blindness. Although it is currently the leading cause of blindness in Japan, the cause is unknown, accurate diagnosis, and effective treatment. And there is no prevention. Among Japanese, normal-tension glaucoma (NTG), which develops glaucoma even though the intraocular pressure is in the normal range, is the most common, and the frequency is about 4% over 40 years of age. Since NTG has a normal range of intraocular pressure, it is difficult to detect by intraocular pressure test, and it is often overlooked by medical examination and normal ophthalmic examination. Because optic nerve damage does not recover, early detection and early treatment are the most important and important in NTG. However, the progression is slow, and the central visual field is damaged late, so that subjective symptoms are poor, and the optic nerve damage progresses without the person's knowledge.

  The search for susceptibility genes for normal-tension glaucoma disease has been carried out, but no gene linked to the disease has yet been found. Several candidate gene analyzes have been undertaken. Myocilin (Non-patent Documents 1-15), optineurin (Non-patent Documents 16-30), and optic atrophy 1 (Non-patent Documents 31-41), which are strongly suspected to be associated with glaucoma, are each energetically analyzed. However, the relationship with NTG is not clear. In addition, the analysis of genes considered as candidates is also underway, but it has only reached a suggestion of association.

  Nakamura et al. Conducted 17 SNP (single nucleotide polymorphism) analysis with the aim of identifying disease-susceptibility genes for normal-tension glaucoma and reported that 17 genes with an association of p <0.000001 were found. Patent Document 42). However, the biologically significant difference is p <0.05, and the p value is an index for determining the presence or absence of association. In particular, when performing a diagnosis using SNP, the level of the value does not indicate only sensitivity and accuracy. For example, identification of SNPs with significant differences in GWAS, alone or in combination, can indicate a high or low risk, and more patients can be confirmed. There are cases where various types can be classified. SNPs that are effective by diagnosis are SNPs that show a significant difference when similar analysis is performed on different groups instead of one group consisting of a patient group and a healthy group.

  An object of the present invention is to find a normal-tension glaucoma disease susceptibility gene effective for diagnosis of normal-tension glaucoma and to provide a method for using the gene.

As a result of comprehensive analysis and comparison of SNPs in the entire genome for both normal-tension glaucoma patient populations and normal populations, the present inventor found that there are differences in multiple SNPs, of which 53 SNPs were clustered The results were found to be good. Furthermore, of the 53 SNPs, we found 4 SNPs that are particularly effective in diagnosing normal-tension glaucoma.
The present invention has been completed based on these findings.

The gist of the present invention is as follows.
(1) The nucleotide of the polymorphic site of the single nucleotide polymorphism international number rs3213787 (the 501st base in the nucleotide sequence of SEQ ID NO: 1 present in the region containing SRBD1 of human chromosome 2), the single nucleotide polymorphism international number rs735860 (The 501st base in the nucleotide sequence of SEQ ID NO: 2 present in the region containing ELOVL5 of human chromosome 6), the base of the polymorphic site of the single nucleotide polymorphism international number rs4412249 (human 6th The base of the polymorphic site of the single nucleotide polymorphism international number rs2763979 (present in HSPA1B of human chromosome 6) in the nucleotide sequence of SEQ ID NO: 3 present in the region containing GMDS of the chromosome P-value (indicator of significant difference between normal-tension glaucoma patients and healthy individuals) in the gene region where the polymorphic site is present is less than 0.05 Other polymorphic parts A method for examining normal-tension glaucoma, comprising identifying a base of at least one polymorphic site selected from the group consisting of bases at a position.
(2) The p-value (indicator of significant difference between normal-tension glaucoma patients and healthy subjects) within the gene region where the polymorphic sites of the single nucleotide polymorphism international numbers rs3213787, rs735860, rs4412249 and rs2763979 are present is less than 0.05 The method according to (1), wherein the other polymorphism is a polymorphism in linkage disequilibrium with the polymorphism of the single nucleotide polymorphism international number rs3213787, rs735860, rs4412249 or rs2763979.
(3) The polymorphism in linkage disequilibrium with the polymorphism of the single nucleotide polymorphism international number rs3213787, rs735860, rs4412249 or rs2763979 is in the same LD block as the single nucleotide polymorphism international number rs3213787, rs735860, rs4412249 or rs2763979 The method according to (2), which is polymorphic.
(4)
A reagent for examining normal-tension glaucoma, comprising at least one component selected from the group consisting of the following components (a) and (b):
(a) the nucleotide of the polymorphic site of the single nucleotide polymorphism international number rs3213787 (the 501st base in the nucleotide sequence of SEQ ID NO: 1 present in the region containing SRBD1 of human chromosome 2), the single nucleotide polymorphism international number rs735860 (The 501st base in the nucleotide sequence of SEQ ID NO: 2 present in the region containing ELOVL5 of human chromosome 6), the base of the polymorphic site of the single nucleotide polymorphism international number rs4412249 (human 6th The base of the polymorphic site of the single nucleotide polymorphism international number rs2763979 (present in HSPA1B of human chromosome 6) P-value (indicator of significant difference between normal-tension glaucoma patients and healthy individuals) in the gene region where the polymorphic site is present is less than 0.05 Other polymorphic sites Primers capable of amplifying a region containing at least one polymorphic site nucleotide is selected from the group consisting of bases
(b) single nucleotide polymorphism international number rs3213787 polymorphic site base (base 501 in nucleotide sequence of SEQ ID NO: 1 present in the region containing SRBD1 of human chromosome 2), single nucleotide polymorphism international number rs735860 (The 501st base in the nucleotide sequence of SEQ ID NO: 2 present in the region containing ELOVL5 of human chromosome 6), the base of the polymorphic site of the single nucleotide polymorphism international number rs4412249 (human 6th The base of the polymorphic site of the single nucleotide polymorphism international number rs2763979 (present in HSPA1B of human chromosome 6) P-value (indicator of significant difference between normal-tension glaucoma patients and healthy individuals) in the gene region where the polymorphic site is present is less than 0.05 Other polymorphic sites At least one polymorphic probe (5) that is capable of hybridizing to a region containing the site of the base probe is immobilized on a solid phase (4) The reagent according selected from the group consisting of bases.
(6) A normal-tension glaucoma test kit comprising the reagent according to (4) or (5).

According to the present invention, normal-tension glaucoma can be more accurately diagnosed. Patients who have already developed a definitive diagnosis, and can be actively treated. In addition, it is possible to predict the onset of unaffected individuals, and it is recommended to conduct tests frequently, which can lead to early detection.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2010-120758, which is the basis of the priority of the present application.

  Hereinafter, embodiments of the present invention will be described in more detail.

  The present invention relates to the base of the polymorphic site of the single nucleotide polymorphism international number rs3213787 (the 501st base in the nucleotide sequence of SEQ ID NO: 1 present in the region containing SRBD1 of human chromosome 2), the single nucleotide polymorphism international number The base of the polymorphic site of rs735860 (the 501st base in the nucleotide sequence of SEQ ID NO: 2 present in the region containing ELOVL5 of human chromosome 6), the base of the polymorphic site of the single nucleotide polymorphism international number rs4412249 (human first The base of the polymorphic site of single nucleotide polymorphism international number rs2763979 (present in the region containing HSPA1B of human chromosome 6), nucleotide number 501 in the nucleotide sequence of SEQ ID NO: 3 present in the region containing GMDS of chromosome 6 P-value (index of significant difference between normal-tension glaucoma patients and healthy individuals) in the gene region where the polymorphic site is present is less than 0.05 Other many A method for examining normal-tension glaucoma, comprising identifying at least one polymorphic site base selected from the group consisting of bases of type sites.

  Other p-values (significant differences between normal-tension glaucoma patients and healthy individuals) within the gene region where the polymorphic sites of single nucleotide polymorphism international numbers rs3213787, rs735860, rs4412249 and rs2763979 exist are less than 0.05 The polymorphism may be a polymorphism in linkage disequilibrium with the polymorphism of the single nucleotide polymorphism international number rs3213787, rs735860, rs4412249 or rs2763979.

  The polymorphism in linkage disequilibrium with the polymorphism of the single nucleotide polymorphism international number rs3213787, rs735860, rs4412249 or rs2763979 is a polymorphism within the LD block of the single nucleotide polymorphism international number rs3213787, rs735860, rs4412249 or rs2763979 Good.

  In the present specification, a single nucleotide polymorphism (SNP) is indicated by an rs number which is a reference SNP ID number in dbSNP which is an NCBI SNP database. The base positions are based on NCBI's genome database, build36.

  In the present specification, “normal tension glaucoma examination” is an examination for determining whether or not a subject has a high or low possibility of having normal tension glaucoma. Tests to make a definitive diagnosis are included.

rs3213787 is a polymorphism of adenine (A) / guanine (G) at the 45500328th base on human chromosome 2. If the base at this site is A, it may have normal tension glaucoma Determined to have high or normal pressure glaucoma.
rs735860 is a polymorphism of thymine (T) / cytosine (C) in the base at position 5331077 on human chromosome 6. If the base at this site is C, there is a possibility of suffering from normal-tension glaucoma. Determined to have high or normal pressure glaucoma.
rs4412249 is a polymorphism of guanine (G) / adenine (A) at the 1947050th base on human chromosome 6. If the base at this site is A, there is a possibility of suffering from normal tension glaucoma. Determined to have high or normal pressure glaucoma.
rs2763979 is a polymorphism of cytosine (C) / thymine (T) at position 31902571 on human chromosome 6. If the base at this site is C, there is a possibility of suffering from normal-tension glaucoma. Determined to have high or normal pressure glaucoma.
The SNP to be identified may be one kind or a combination of two or more, but preferably includes either rs3213787 or rs735860, or both rs3213787 and rs735860, and in addition, rs4412249 and / or rs2763979 May be included. Moreover, the sense strand of the gene may be analyzed, or the antisense strand may be analyzed.
In the present invention, the base to be identified is not limited to the above SNP, and may be a base at a polymorphic site in linkage disequilibrium with the above SNP.

Other p-values (significant differences between normal-tension glaucoma patients and healthy individuals) within the gene region where the polymorphic sites of single nucleotide polymorphism international numbers rs3213787, rs735860, rs4412249 and rs2763979 exist are less than 0.05 Examples of the polymorphism include polymorphisms whose p-value (an index of a significant difference between normal-tension glaucoma patients and healthy subjects) is less than 0.05 in Tables A, B, C, and D described later.
Table A shows the polymorphic site and p-value within the gene region (SRBD1) where the single nucleotide polymorphism international number rs3213787 exists.

(Table A)

(Table A) (continued)

Table B shows the site and p-value of the polymorphism in the gene region (ELOVL5) where the single nucleotide polymorphism international number rs735860 exists.
(Table B)

(Table B) (continued)

Table C shows the polymorphic site and p-value in the gene region (GMDS) where the single nucleotide polymorphism international number rs4412249 exists.

(Table C)

(Table C) (continued)

(Table C) (continued)

(Table C) (continued)

(Table C) (continued)

Table D shows the site and p-value of the polymorphism in the gene region (HSPA1B) where the single nucleotide polymorphism international number rs2763979 is present.

(Table D)

(Table D) (continued)

A large D 'between SNPs is considered to be in linkage disequilibrium (Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005; 21 (2) Gabriel SB, Schaffner SF, Nguyen H, et al. The structure of haplotype blocks in the human genome. Science; 2002; 296 (5576): 2225-2229.). Accordingly, polymorphisms that are in linkage disequilibrium with the polymorphisms of the single nucleotide polymorphism international numbers rs3213787, rs735860, rs4412249 or rs2763979 are, for example, polymorphisms having a larger D ′ between these SNPs.

  LD block is the method of Gabriel et al. Using Haploview software (Barrett JC, Fry B, Maller J, Daly MJ.Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005; 21 (2): 263-265.) (Gabriel SB, Schaffner SF, Nguyen H, et al. The structure of haplotype blocks in the human genome. Science. 2002; 296 (5576): 2225-2229.).

  The polymorphisms in the LD blocks of the single nucleotide polymorphism international numbers rs3213787, rs735860, rs4412249, and rs2763979 are the gray bands around the lead SNP shown in bold bold in Tables A, B, C, and D above. Although the following can be illustrated, it is not necessarily limited to these.

Polymorphisms in the LD block of the single nucleotide polymorphism international number rs3213787: rs11888802, rs11678872, rs2343468, rs13003019, rs6760244, rs12471726, rs2081297, rs10460504, rs7562458, rs4953230, rs4952763, rs6719211, rs1014454, rs3755070, rs3213rs, rs10291, rs4455206, rs3755076, rs3770254, rs11125017, rs3770264, rs10205197, rs10427308, rs17033745, rs3770278, rs17322370, rs6736954, rs10427402, rs4476395, rs13023749, rs17033801, rs755652, rs748573, rs127, rs6710581, rs127 rs7596942 and rs7565326
Polymorphisms in the LD block of the single nucleotide polymorphism international number rs735860: rs2562898, rs735860, rs715441, rs2057024, rs1429146, rs9463895, rs2235723, rs1346603, rs9474476, rs2294867, rs9349660, rs974323, rs6909592, rs9397520, rs9395854, rs209485, rs939856 rs7747926, rs7738788, rs209500, rs9357760, rs9370196, rs209512, rs209517, and rs9370201
Polymorphisms in the LD block of the single nucleotide polymorphism international number rs4412249: rs7772545, rs6596866, rs4959621, rs4412249, rs4367413, rs11242729, rs6925298, rs12212324, rs9392353, rs11242730, rs4580917, rs6910252, rs11242733, rs9503086, rs11242937, rs476594 rs676015, rs2026446 and rs4959631
Polymorphisms in the LD block of single nucleotide polymorphism international number rs2763979: rs707918, rs805292, rs2299851, rs707939, rs707937, rs480092, rs2075800, rs2763979 and rs11965547

  The “polymorphic site” in the test method of the present invention refers to a gene ORF, a region that controls gene expression (for example, a promoter region, an enhancer region, etc.), a gene intron, or a linkage disequilibrium with these genes. It can exist in some area before and after. Examples of polymorphisms include single nucleotide polymorphisms, polymorphisms in which one to several tens of bases (sometimes several thousand bases) are substituted, deleted, inserted, transferred, or inverted. There is no particular limitation.

  In the inspection method of the present invention, the identification of the base at the polymorphic site (that is, the determination of the base type) can be performed by a known single nucleotide polymorphism analysis method. Examples of single nucleotide polymorphism analysis methods include, but are not limited to, sequence analysis, PCR, PCR-SSCP, hybridization, HRM method, RFLP method and the like.

  In order to identify the base of the polymorphic site, genomic DNA may be extracted from a biological sample of the subject. Biological samples include, for example, the subject's blood, skin, oral mucosa, tissues or cells collected or excised by surgery, body fluids collected for the purpose of examination (saliva, lymph, airway mucosa, semen, sweat, urine, etc.) ) Etc. As the biological sample, leukocytes or mononuclear cells separated from peripheral blood are preferable. Genomic DNA can be extracted from a biological sample using a commercially available DNA extraction kit. Then, if necessary, DNA containing the polymorphic site is isolated. The DNA can be isolated by PCR or the like using genomic DNA or RNA as a template, using a primer capable of hybridizing to DNA containing a polymorphic site.

The present invention also provides a reagent for examining normal-tension glaucoma, comprising at least one component selected from the group consisting of the following components (a) and (b).
(a) the nucleotide of the polymorphic site of the single nucleotide polymorphism international number rs3213787 (the 501st base in the nucleotide sequence of SEQ ID NO: 1 present in the region containing SRBD1 of human chromosome 2), the single nucleotide polymorphism international number rs735860 (The 501st base in the nucleotide sequence of SEQ ID NO: 2 present in the region containing ELOVL5 of human chromosome 6), the base of the polymorphic site of the single nucleotide polymorphism international number rs4412249 (human 6th The base of the polymorphic site of the single nucleotide polymorphism international number rs2763979 (present in HSPA1B of human chromosome 6) in the nucleotide sequence of SEQ ID NO: 3 present in the region containing GMDS of the chromosome P-value (indicator of significant difference between normal-tension glaucoma patients and healthy individuals) in the gene region where the polymorphic site is present is less than 0.05 Other polymorphic sites Primers capable of amplifying a region containing at least one polymorphic site nucleotide is selected from the group consisting of bases
(b) single nucleotide polymorphism international number rs3213787 polymorphic site base (base 501 in nucleotide sequence of SEQ ID NO: 1 present in the region containing SRBD1 of human chromosome 2), single nucleotide polymorphism international number rs735860 (The 501st base in the nucleotide sequence of SEQ ID NO: 2 present in the region containing ELOVL5 of human chromosome 6), the base of the polymorphic site of the single nucleotide polymorphism international number rs4412249 (human 6th The base of the polymorphic site of the single nucleotide polymorphism international number rs2763979 (present in HSPA1B of human chromosome 6) in the nucleotide sequence of SEQ ID NO: 3 present in the region containing GMDS of the chromosome P-value (indicator of significant difference between normal-tension glaucoma patients and healthy individuals) in the gene region where the polymorphic site is present is less than 0.05 Other polymorphic sites Probe capable of hybridizing to a region containing bases of at least one polymorphic site selected from the group consisting of bases Polymorphic site of single nucleotide polymorphism international number rs3213787, polymorphism of single nucleotide polymorphism international number rs735860 Site, polymorphic site of single nucleotide polymorphism international number rs4412249, polymorphic site of single nucleotide polymorphism international number rs2763979, and p-value in the gene region where these polymorphic sites exist (normal tension glaucoma patients and healthy Other polymorphic sites having an index of significant difference between individuals of less than 0.05 are as described above.
Furthermore, the present invention provides a normal-tension glaucoma test kit comprising the above-described reagent.

  The primer and probe that are components of the reagent of the present invention may be an oligonucleotide having a chain length of at least 15 nucleotides. When the oligonucleotide is used as a primer, the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least a part of the DNA containing the polymorphic site. The length of the DNA that can be amplified by the primer is usually 15 to 1000 bp, preferably 20 to 500 bp, more preferably 20 to 200 bp. Moreover, when using this oligonucleotide as a probe, the length is 7 bp-500 bp normally, Preferably it is 8 bp-500 bp. The probe is not particularly limited as long as it can hybridize with the DNA containing the polymorphic site. The length of DNA to which the probe can hybridize is usually 16 to 500 bp, preferably 20 to 200 bp, more preferably 20 to 50 bp.

  In the present invention, a primer capable of amplifying a region containing a polymorphic site is preferably one that can initiate complementary strand synthesis toward the polymorphic site using DNA containing the polymorphic site as a template.

  In addition to the base sequence complementary to the base sequence of the region containing the polymorphic site, an arbitrary base sequence can be added to the primer. For example, in a primer for a polymorphism analysis method using a type IIs restriction enzyme, a primer to which a recognition sequence for a type IIs restriction enzyme is added is used. Furthermore, the primer may be modified. For example, a primer labeled with a fluorescent substance or a binding affinity substance such as biotin or digoxin may be used.

  In the present invention, the probe that can hybridize to the region containing the polymorphic site may be any probe that can hybridize to the polynucleotide having the base sequence of the region containing the polymorphic site. Those that specifically hybridize to DNA having the base sequence of the region to be included are preferred. Here, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, In the condition described in the second edition 1989), it means that cross-hybridization does not occur significantly with DNA other than DNA having the base sequence of the region containing the polymorphic site. More specifically, a probe containing a polymorphic site in the base sequence of the probe is preferable. Alternatively, depending on the base analysis method at the polymorphic site, the probe may be designed so that the end of the probe corresponds to a base adjacent to the polymorphic site. Therefore, although the polymorphic site is not included in the base sequence of the probe itself, a probe including a base sequence complementary to the region adjacent to the polymorphic site can also be shown as a desirable probe in the present invention.

  The probe is allowed to modify the base sequence, add the base sequence, or modify the same as the primer. For example, a probe used for the Invader method is added with a base sequence unrelated to the genome constituting the flap. Such a probe is also included in the probe of the present invention as long as it hybridizes to a region containing a polymorphic site. The base sequence constituting the probe of the present invention can be designed according to the analysis method based on the base sequence of the DNA region surrounding the polymorphic site of the present invention in the genome.

  A person skilled in the art can design primers and probes according to the analysis method based on the base sequence information about the surrounding DNA region including the polymorphic site. The base sequences constituting the primers and probes can be modified as appropriate as well as the base sequences that are completely complementary to the genomic base sequences.

  Primers and probes can be synthesized by any method based on the base sequences constituting them. A technique for synthesizing an oligonucleotide having the base sequence based on the given base sequence is known. Furthermore, in the synthesis of the oligonucleotide, any modification can be introduced into the oligonucleotide using a nucleotide derivative modified with a fluorescent dye or biotin. Alternatively, a method of binding a fluorescent dye or the like to a synthesized oligonucleotide is also known.

  The probe may be immobilized on a solid phase (DNA array). In the DNA array, sample DNA (or RNA) is hybridized to a large number of probes arranged on the same plane, and the hybridization to each probe is detected by scanning the plane. Since responses to many probes can be observed simultaneously, for example, a DNA array is useful for analyzing a large number of polymorphic sites simultaneously. Examples of nucleotide immobilization (array) methods include arrays based on oligonucleotides developed by Affymetrix. In an oligonucleotide array, oligonucleotides are usually synthesized in situ. For example, in situ synthesis methods of oligonucleotides by lithography method (Affymetrix), inkjet method (Agilent), bead array method (Illumina), etc. are known.

  The oligonucleotide is composed of a base sequence complementary to a region containing a polymorphic site to be detected. The length of the nucleotide probe to be bound to the substrate is usually 10 to 100 bp, preferably 10 to 50 bp, more preferably 15 to 25 bp when the oligonucleotide is immobilized.

  A sample for SNP detection by the DNA array method can be prepared by a method well known to those skilled in the art based on a biological sample collected from a subject. The biological sample is not particularly limited. For example, a DNA sample can be prepared from genomic DNA extracted from tissues or cells of peripheral blood leukocytes, skin, oral mucosa, etc., tears, saliva, urine, feces or hair of the subject. A specific region of genomic DNA is amplified using a primer for amplifying a region containing a polymorphic site to be determined. At this time, a plurality of regions can be simultaneously amplified by the multiplex PCR method. The multiplex PCR method is a PCR method using a plurality of primer sets in the same reaction solution. When analyzing multiple polymorphic sites, the multiplex PCR method is useful.

  In general, in the DNA array method, a DNA sample is amplified by a PCR method and an amplification product is labeled. A labeled primer is used for labeling the amplification product. For example, genomic DNA is first amplified by PCR using a primer set specific to the region containing the polymorphic site. Next, biotin-labeled DNA is synthesized by a labeling PCR method using a biotin-labeled primer. The biotin-labeled DNA synthesized in this way is hybridized to the oligonucleotide probe on the chip. The hybridization reaction solution and reaction conditions can be appropriately adjusted according to conditions such as the length of the nucleotide probe immobilized on the solid phase and the reaction temperature. One skilled in the art can design appropriate hybridization conditions. In order to detect the hybridized DNA, avidin labeled with a fluorescent dye is added. The array is analyzed with a scanner, and the presence or absence of hybridization is confirmed using fluorescence as an index.

  An example of a procedure for carrying out the test method of the present invention using the DNA array method is as follows. After preparing a solid phase on which a DNA and nucleotide probe containing a polymorphic site prepared from a subject are immobilized, The solid phase is contacted. Subsequently, the base species of the polymorphic site is determined by detecting DNA hybridized to the nucleotide probe immobilized on the solid phase.

  As used herein, “solid phase” means a material capable of immobilizing nucleotides. The solid phase is not particularly limited as long as nucleotides can be immobilized, and specific examples include a solid phase containing microplate wells, plastic beads, magnetic particles, a substrate, and the like. As the solid phase, a substrate generally used in DNA array technology can be preferably used. In the present specification, the “substrate” means a plate-like material capable of fixing nucleotides. In the present invention, the nucleotide includes oligonucleotides and polynucleotides.

  In addition to the above method, an allele specific oligonucleotide (ASO) hybridization method can be used to detect a base at a specific site. An allele-specific oligonucleotide (ASO) is composed of a base sequence that hybridizes to a region where a polymorphic site to be detected exists. When ASO is hybridized to sample DNA, the hybridization efficiency decreases if a mismatch occurs at the polymorphic site due to the polymorphism. Mismatches can be detected by Southern blotting or a method that uses the property of quenching by intercalating a special fluorescent reagent into the hybrid gap. Mismatches can also be detected by the ribonuclease A mismatch cleavage method.

  The reagents and kits of the present invention can contain various enzymes, enzyme substrates, buffers, and the like depending on the base identification method. Examples of the enzyme include enzymes necessary for the various analysis methods exemplified as the base identification method, such as DNA polymerase, DNA ligase, or IIs restriction enzyme. As the buffer solution, a buffer solution suitable for maintaining the activity of the enzyme used for these analyzes is appropriately selected. Furthermore, as the enzyme substrate, for example, a substrate for complementary strand synthesis is used.

  Furthermore, the reagent and kit of the present invention can be accompanied by a control in which the base at the polymorphic site is clear. As a control, genomic DNA or a fragment of genomic DNA in which the base type of the polymorphic site is known in advance can be used. Genomic DNA extracted from cells may be attached as a control, or a cell or a fraction of cells may be attached as a control, and a user may extract genomic DNA therefrom. If a cell is used as a control, the result of the control can prove that the genomic DNA extraction operation was performed correctly. Alternatively, DNA comprising a base sequence containing a polymorphic site can be used as a control. Specifically, a YAC vector or a BAC vector containing a genome-derived DNA whose base type at the polymorphic site has been clarified may be used as a control. Alternatively, a vector in which only tens to hundreds of bp corresponding to the polymorphic site are excised and inserted can be used as a control.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.

[Example 1]
NTG patients and healthy subjects collected blood leukocytes from NTG patients who met strict criteria such as age, intraocular pressure, refractive error, etc. with the permission of the Yokohama City University School of Medicine and Ethics Committee.

  NTG's diagnostic criteria follow the criteria of the Japan Glaucoma Society.

  The primary gene was 305 patients and 355 healthy subjects, which were narrowed down to 53 gene regions. As a subsequent replication, we identified 214 SNPs and 257 healthy subjects, and identified 4SNPs expected to be highly effective. These four genes are shown in the table, and in addition to the results of primary and replication, values calculated for all specimens are shown.

Genotyping was performed using GeneChip Human Mapping 500K Array Set (Affymetrix) according to a standard protocol recommended by Affymetrix.
For analysis of GWAS including statistical analysis, Helixtree SVS 7 (Golden Herix. Inc. Bozeman, Montana, USA) and Haploview v4.1 were used.
Haploview v4.1 was also used to estimate the LD structure at the target position.
The results are shown in the table below. rs3213787, rs735860, rs4412249, and rs2763979 showed significant correlation with NTG in both GWAS and Replication, and were proved to be effective SNPs for the diagnosis of NTG.

The following table shows the new results of increasing the number of patients and healthy individuals (added data for 46 patients and 46 healthy people to the above data). Additional patients and healthy individuals showed similar allele frequency trends.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

  The present invention can be used for life science, medicine, ophthalmology and diagnosis.

<SEQ ID NO: 1>
SEQ ID NO: 1 shows a 1000-base long base sequence containing the polymorphic site of rs3213787 at the 501st (r = A / G).
<SEQ ID NO: 2>
SEQ ID NO: 2 shows a 1000-base long base sequence containing the polymorphic site of rs735860 at the 501st (y = C / T).
<SEQ ID NO: 3>
SEQ ID NO: 3 shows a 1000-base long base sequence containing the polymorphic site of rs4412249 at the 501st (r = A / G).
<SEQ ID NO: 4>
SEQ ID NO: 4 shows a 1000-base long base sequence containing the polymorphic site of rs2763979 at the 501st (y = C / T).

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Claims (6)

Polynucleotide of polymorphic site of single nucleotide polymorphism international number rs3213787 (base 501 in nucleotide sequence of SEQ ID NO: 1 present in the region containing SRBD1 of human chromosome 2), polymorphism of single nucleotide polymorphism international number rs735860 The base of the site (base 501 in the nucleotide sequence of SEQ ID NO: 2 present in the region containing ELOVL5 of human chromosome 6), the base of the polymorphic site of single nucleotide polymorphism international number rs4412249 (GMDS of human chromosome 6) 501 base in the nucleotide sequence of SEQ ID NO: 3 present in the region containing), the base of the polymorphic site of the single nucleotide polymorphism international number rs2763979 (SEQ ID NO: 4 present in the region containing HSPA1B of human chromosome 6) 501 in the nucleotide sequence of), and other polymorphisms whose p-value (indicator of significant difference between normal-tension glaucoma patients and healthy individuals) in the gene region where the polymorphic site is less than 0.05 Mold site salt A method for examining normal tension glaucoma, comprising identifying a base of at least one polymorphic site selected from the group consisting of groups. Other p-values (significant differences between normal-tension glaucoma patients and healthy individuals) within the gene region where the polymorphic sites of single nucleotide polymorphism international numbers rs3213787, rs735860, rs4412249 and rs2763979 exist are less than 0.05 The method according to claim 1, wherein the polymorphism is a polymorphism in linkage disequilibrium with the polymorphism of the single nucleotide polymorphism international number rs3213787, rs735860, rs4412249 or rs2763979. The polymorphism in linkage disequilibrium with the polymorphism of the single nucleotide polymorphism international number rs3213787, rs735860, rs4412249 or rs2763979 is a polymorphism within the LD block of the single nucleotide polymorphism international number rs3213787, rs735860, rs4412249 or rs2763979 Item 3. The method according to Item 2. A reagent for examining normal-tension glaucoma, comprising at least one component selected from the group consisting of the following components (a) and (b):
(a) the nucleotide of the polymorphic site of the single nucleotide polymorphism international number rs3213787 (the 501st base in the nucleotide sequence of SEQ ID NO: 1 present in the region containing SRBD1 of human chromosome 2), the single nucleotide polymorphism international number rs735860 (The 501st base in the nucleotide sequence of SEQ ID NO: 2 present in the region containing ELOVL5 of human chromosome 6), the base of the polymorphic site of the single nucleotide polymorphism international number rs4412249 (human 6th The base of the polymorphic site of the single nucleotide polymorphism international number rs2763979 (present in HSPA1B of human chromosome 6) in the nucleotide sequence of SEQ ID NO: 3 present in the region containing GMDS of the chromosome P-value (indicator of significant difference between normal-tension glaucoma patients and healthy individuals) in the gene region where the polymorphic site is present is less than 0.05 Other polymorphic sites Primers capable of amplifying a region containing at least one polymorphic site nucleotide is selected from the group consisting of bases
(b) single nucleotide polymorphism international number rs3213787 polymorphic site base (base 501 in nucleotide sequence of SEQ ID NO: 1 present in the region containing SRBD1 of human chromosome 2), single nucleotide polymorphism international number rs735860 (The 501st base in the nucleotide sequence of SEQ ID NO: 2 present in the region containing ELOVL5 of human chromosome 6), the base of the polymorphic site of the single nucleotide polymorphism international number rs4412249 (human 6th The base of the polymorphic site of the single nucleotide polymorphism international number rs2763979 (present in HSPA1B of human chromosome 6) in the nucleotide sequence of SEQ ID NO: 3 present in the region containing GMDS of the chromosome P-value (indicator of significant difference between normal-tension glaucoma patients and healthy individuals) in the gene region where the polymorphic site is present is less than 0.05 Other polymorphic sites Probe capable of hybridizing to a region including at least one polymorphic site nucleotide is selected from the group consisting of bases The reagent according to claim 4, wherein the probe is fixed to a solid phase. A test kit for normal-tension glaucoma, comprising the reagent according to claim 4 or 5.