KR20130041767A - Normal-tension glaucoma susceptibility gene and method for using the same - Google Patents

Normal-tension glaucoma susceptibility gene and method for using the same Download PDF

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KR20130041767A
KR20130041767A KR1020127023847A KR20127023847A KR20130041767A KR 20130041767 A KR20130041767 A KR 20130041767A KR 1020127023847 A KR1020127023847 A KR 1020127023847A KR 20127023847 A KR20127023847 A KR 20127023847A KR 20130041767 A KR20130041767 A KR 20130041767A
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노부히사 미즈키
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가부시끼가이샤 메니콘
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Priority to PCT/JP2011/058164 priority patent/WO2011148715A1/en
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    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • C12Q2600/00Oligonucleotides characterized by their use
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Abstract

Provided are normal intraocular glaucoma disease susceptibility genes and methods of using the same.
Among the polymorphic sites that have been comprehensively analyzed and compared with the single nucleotide polymorphisms in the whole genome of a large number of patients with normal-tension glaucoma and healthy people, the polymorphic sites of the single nucleotide polymorphisms international numbers rs3213787, rs735860, rs4412249 and rs2763979 It was found to be valid for diagnosis.

Description

Normal tension glaucoma disease susceptibility gene and its use {NORMAL-TENSION GLAUCOMA SUSCEPTIBILITY GENE AND METHOD FOR USING THE SAME}
The present invention relates to normal intraocular glaucoma disease susceptibility genes and their use.
Glaucoma is an advanced refractory disease that causes the optic nerve to fail due to an increase in intraocular pressure above healthy IOP that can maintain normal visual function. When left unattended, visual field narrowing progresses and blindness occurs, and although it occupies the first place in the cause of blindness in Japan at present, the cause is unknown and there is no accurate diagnosis, effective treatment, and preventive method. Among the Japanese, the intraocular pressure is the highest in the normal range, but the most common is normal intraocular glaucoma (NTG) that develops glaucoma, the frequency is about 4% over 40 years old. Since NTG is in the normal range of intraocular pressure, it is difficult to detect it in an intraocular pressure test, and it tends to be overlooked in a medical examination or an ordinary ophthalmologic examination. Because optic nerve disorders do not recover, early detection and early treatment are the most important and important in NTG. However, the progress is slow, and the central field of vision is impaired in the late stages, so the subjective symptoms are insufficient, and optic nerve injury proceeds without knowledge of the person.
The search for susceptibility genes to normal-tension glaucoma disease has been conducted, but no genes linked to the disease have yet been found. Several candidate gene analyzes have been conducted. The analysis of myocilin (non-patent document 1-15), optineurin (non-patent document 16-30), and optic atrophy 1 (non-patent document 31-41) genes, which are strongly suspected to be associated with glaucoma, has been performed energetically. However, the relationship with NTG is not clear. In addition, interpretation of genes considered to be candidates is also in progress, and only related suggestions have been reached.
Nakamura et al. Conducted SNP (one-nucleotide polymorphism) analysis to identify disease-sensitive genes in normal-tension glaucoma, and reported that 17 genes with an association of p <0.000001 were found (Non-Patent Document 42). ). However, the biological difference is p <0.05 and the p value is an indicator for determining the presence or absence of related. In particular, when the diagnosis is performed using SNP, the high and low values do not only indicate sensitivity and precision. For example, by identifying single or combination of SNPs with significant differences in GWAS, which can suggest a higher or lower risk, more confirmed patients can be diagnosed, and the various forms included in the disease. There is a case where can be classified. An effective SNP for diagnosis is not a group unit consisting of a patient group and a healthy human group, but an SNP that recognizes a significant difference even when the same interpretation is performed in different groups.
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An object of the present invention is to find a normal intraocular glaucoma disease susceptibility gene effective for diagnosing normal intraocular glaucoma and to provide a method of using the same.
The present inventors have analyzed and compared the SNPs in the whole genome in a group of patients with normal-tension glaucoma and a healthy human group, and found that there are differences in a plurality of SNPs, and among them 53 SNPs have good cluster analysis results. Figured out. Of the 53 SNPs, four SNPs that were particularly effective for the diagnosis of normal intraocular glaucoma were found. The present invention has been completed based on these findings.
The gist of the present invention is as follows.
(1) The base of the polymorphic site of the single-nucleotide polymorphic international number rs3213787 (the 501st base in the nucleotide sequence of SEQ ID NO: 1 present in the region containing SRBD1 of the human second chromosome), and the mononucleotide polymorphic international number rs735860 of Base of polymorphic site (501st base in nucleotide sequence of SEQ ID NO: 2 present in a region containing ELOVL5 of human 6th chromosome), base of polymorphic site of mononucleotide polymorphic international number rs4412249 (of human 6th chromosome) 501st base in nucleotide sequence of SEQ ID NO: 3 present in a region including GMDS), base of the polymorphic region of the single-nucleotide polymorphism international number rs2763979 (SEQ ID NO: present in the region including HSPA1B of the human sixth chromosome) The 501st base in the nucleotide sequence of 4), and p in the gene region in which these polymorphic sites exist NTG inspection method, comprising: identifying at least one nucleotide of the polymorphic site that is (normal tension glaucoma patients and healthy individuals significantly different index of between a) selected from the group consisting of a base of 0.05 of less than other polymorphic sites.
(2) Other polymorphisms with a p value (indicator of significant difference between normal tension glaucoma patients and healthy persons) within the gene region in which the polymorphic sites of the single-base polymorphisms international numbers rs3213787, rs735860, rs4412249 and rs2763979 are present are 1 The method according to (1), which is a polymorph in polymorphism and chain imbalance of base polymorphic international numbers rs3213787, rs735860, rs4412249, or rs2763979.
(3) The method described in (2), wherein the polymorph in polymorphism and chain imbalance of the single base polymorphic international number rs3213787, rs735860, rs4412249 or rs2763979 is a polymorph in the same LD block as the one-base polymorphic international number rs3213787, rs735860, rs4412249 or rs2763979 .
(4) A reagent for performing a normal intraocular glaucoma test, comprising at least one component selected from the group consisting of the following components (a) and (b).
(a) 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 the human second chromosome), and the single-nucleotide polymorphism international number rs735860 of Base of polymorphic site (501st base in nucleotide sequence of SEQ ID NO: 2 present in a region containing ELOVL5 of human 6th chromosome), base of polymorphic site of mononucleotide polymorphic international number rs4412249 (of human 6th chromosome) 501st base in nucleotide sequence of SEQ ID NO: 3 present in a region including GMDS), base of the polymorphic region of the single-nucleotide polymorphism international number rs2763979 (SEQ ID NO: present in the region including HSPA1B of the human sixth chromosome) The 501st base in the nucleotide sequence of 4), and p in the gene region in which the polymorphic site exists A primer capable of amplifying a region comprising a base of at least one polymorphic site selected from the group consisting of bases of other polymorphic sites with a value (indicator of significant difference between normal tension glaucoma patients and healthy persons) of less than 0.05.
(b) the base of the polymorphic site of the single-nucleotide polymorphic international number rs3213787 (the 501st base in the nucleotide sequence of SEQ ID NO: 1 present in the region containing SRBD1 of the human second chromosome), and the single-nucleotide polymorphic international number rs735860 Base of polymorphic site (501st base in nucleotide sequence of SEQ ID NO: 2 present in a region containing ELOVL5 of human 6th chromosome), base of polymorphic site of mononucleotide polymorphic international number rs4412249 (of human 6th chromosome) 501st base in nucleotide sequence of SEQ ID NO: 3 present in a region including GMDS), base of the polymorphic region of the single-nucleotide polymorphism international number rs2763979 (SEQ ID NO: present in the region including HSPA1B of the human sixth chromosome) The 501st base in the nucleotide sequence of 4), and p in the gene region in which the polymorphic site exists Probe capable of hybridizing to a region containing bases of at least one polymorphic site selected from the group consisting of bases of other polymorphic sites with a value (indicator of significant difference between normal tension glaucoma patients and healthy persons) of less than 0.05 .
(5) The reagent according to (4), wherein the probe is fixed to a solid phase.
(6) A normal intraocular glaucoma test kit comprising the reagent according to (4) or (5).
According to the present invention, it becomes possible to diagnose a normal intraocular glaucoma more accurately. Confirmed diagnosis is possible for patients who have already developed, and active treatment can be performed. In addition, it is possible to predict the onset of undeveloped people, and it is recommended to perform the test several times, which may lead to early detection.
This specification includes part or all of the contents as disclosed in the description and / or drawings of Japanese Patent Application, Japanese Patent Application No. 2010-120758, which is a priority document of the present application.
Hereinafter, embodiments of the present invention will be described in more detail.
The present invention relates to a nucleotide sequence of the polymorphic moiety of the single nucleotide polymorphism international number rs3213787 (the 501st base in the nucleotide sequence of SEQ ID NO: 1 present in a region containing SRBD1 of the human second chromosome), Base of polymorphic site (501st base in nucleotide sequence of SEQ ID NO: 2 present in a region containing ELOVL5 of human 6th chromosome), base of polymorphic site of mononucleotide polymorphic international number rs4412249 (of human 6th chromosome) 501st base in nucleotide sequence of SEQ ID NO: 3 present in a region including GMDS), base of the polymorphic region of the single-nucleotide polymorphism international number rs2763979 (SEQ ID NO: present in the region including HSPA1B of the human sixth chromosome) 501st base in nucleotide sequence 4), and in the gene region in which the polymorphic site exists Normal intraocular glaucoma screening comprising identifying bases of at least one polymorphic site selected from the group consisting of bases of other polymorphic sites with a p value (indicator of significant difference between normal tension glaucoma patients and healthy individuals) of less than 0.05. Provide a method.
Other polymorphisms with a p-value (indicative of significant differences between patients with normal-tension glaucoma and healthy people) within the gene region in which the polymorphic sites of the single-base polymorphic international numbers rs3213787, rs735860, rs4412249 and rs2763979 exist are This can be done with the polymorphism in the polymorphism and chain unbalance of the number rs3213787, rs735860, rs4412249 or rs2763979.
Polymorphs in the polymorph and chain unbalance of the single-base polymorphic international number rs3213787, rs735860, rs4412249 or rs2763979 may be polymorphs in the LD block of the single-base polymorphic international number rs3213787, rs735860, rs4412249 or rs2763979.
In the present specification, the single nucleotide polymorphism (SNP) is represented by an rs number which is a reference SNP ID number in dbSNP which is an SNP database of NCBI. The base position is based on the NCBI's genomic database, build36.
In the present specification, the term "normal intraocular glaucoma test" includes a test for determining whether a subject has a high or low likelihood of having normal intraocular glaucoma, and a test for performing a definite diagnosis when the subject has already received normal intraocular glaucoma. do.
rs3213787 is a polymorph of adenine (A) / guanine (G) at the 45500328th base on the human second chromosome, and when the base of this site is A, the possibility of developing normal intraocular glaucoma is high, or normal intraocular pressure It is determined that you have glaucoma.
rs735860 is a polymorph of thymine (T) / cytosine (C) at the 53231077th base on the human 6th chromosome, and when the base of this site is C, the possibility of developing normal intraocular glaucoma is high, or normal intraocular pressure It is determined that you have glaucoma.
rs4412249 is a polymorph of guanine (G) / adenine (A) at the 1947050th base on the human 6th chromosome, and when the base of this site is A, it is highly likely to develop normal intraocular glaucoma or normal intraocular pressure It is determined that you have glaucoma.
rs2763979 is a polymorphism of cytosine (C) / thymine (T) at the 31902571st base on the human 6th chromosome, and when the base of this site is C, there is a high possibility of developing normal intraocular glaucoma or normal intraocular pressure. It is determined that you have glaucoma.
One type of SNP to be identified may be used or a plurality of combinations thereof may be used, but any one of rs3213787 or rs735860, or both rs3213787 and rs735860 may be included, and in addition, rs4412249 and / or rs2763979 may be included. Moreover, you may analyze the sense chain of a gene and may analyze an antisense chain.
In addition, in the present invention, the base to be identified is not limited to the SNP described above, and may be a base of a polymorphic site in the chain unbalance with the SNP.
Other polymorphisms having a p-value (an indicator of significant difference between normal and high-tension glaucoma patients and healthy persons) in the gene region in which the polymorphic sites of the single-base polymorphic international numbers rs3213787, rs735860, rs4412249 and rs2763979 exist are described below. In Tables A, B, C, and D, polymorphisms with p values (indicators of significant differences between normal tension glaucoma patients and healthy people) can be illustrated.
Table A shows the site and p value of the polymorphism in the gene region (SRBD1) where the single nucleotide polymorphism international number rs3213787 is present.
Table A
Figure pct00006
Figure pct00007
Table B shows the sites and p values of the polymorphisms in the gene region (ELOVL5) where the single nucleotide polymorphism international number rs735860 is present.
Table B
Figure pct00008
Figure pct00009
Table C shows the sites and p values of the polymorphisms in the genetic region (GMDS) where the single nucleotide polymorphism international number rs4412249 is present.
Table C
Figure pct00010
Figure pct00011
Figure pct00012
Figure pct00013
Figure pct00014
Table D shows the sites and p values of the polymorphisms in the gene region (HSPA1B) where the single nucleotide polymorphism international number rs2763979 is present.
Table D
Figure pct00015
Figure pct00016
Large D's between SNPs are thought to be in chain imbalance (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). Thus, the polymorph of the single-base polymorphic international number rs3213787, rs735860, rs4412249 or rs2763979 and the polymorphism in the chain unbalance is, for example, a polymorph with a larger D 'between these SNPs.
LD blocks were analyzed using the 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).
Polymorphs in the LD blocks of the single-base polymorphic international numbers rs3213787, rs735860, rs4412249, and rs2763979 include, in Tables A, B, C, and D, described above, as indicated by the gray band around the lead SNP in bold bold: Although it can illustrate, it is not limited to these.
Polymorph in LD block of mononucleotide polymorphic international number rs3213787:
Figure pct00017
Polymorph in LD block of mononucleotide polymorphic international number rs735860:
Figure pct00018
Polymorph in LD block of mononucleotide polymorphic international number rs4412249:
Figure pct00019
Polymorph in LD block of mononucleotide polymorphic international number rs2763979:
Figure pct00020
The "polymorphic site" in the test method of the present invention is a chain of genes in the introns of genes or in the introns of genes in the region which controls gene expression (for example, a promoter region, an enhancer region, etc.) in ORF of a gene. It can exist in the region before and after the imbalance. Examples of the polymorphism include, but are not particularly limited to, one-base polymorphisms and polymorphisms in which one to several tens of bases (sometimes thousands of bases) are substituted, deleted, inserted, transferred or reversed.
In the test method of the present invention, identification of the base of the polymorphic site (that is, determination of the base species) can be carried out by a known one-base polymorphic analysis method. As the mononucleotide polymorphism analysis method, sequence analysis, PCR, PCR-SSCP, hybridization, HRM method, RFLP method, and the like can be exemplified, but are not limited thereto.
In order to identify the base of a polymorphic site, genomic DNA may be extracted from the biological sample of a subject. The biological sample may be, for example, blood, skin, oral mucosa of a subject, tissue or cells collected or excised by surgery, or a body fluid (saliva, lymph, airway mucosa, semen, sweat, urine, etc.) collected for the purpose of testing. ). As the biological sample, leukocytes or monocytes isolated from peripheral blood are preferable. A commercial DNA extraction kit can be used to extract genomic DNA from biological samples. Then, if necessary, the DNA containing the polymorphic site is isolated. Isolation of the DNA can be performed by PCR using genomic DNA or RNA as a template, using primers capable of hybridizing to DNA containing polymorphic sites.
In addition, the present invention provides a reagent for a normal intraocular glaucoma test, comprising at least one component selected from the group consisting of the following components (a) and (b).
(a) 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 the human second chromosome), and the single-nucleotide polymorphism international number rs735860 of Base of polymorphic site (501st base in nucleotide sequence of SEQ ID NO: 2 present in a region containing ELOVL5 of human 6th chromosome), base of polymorphic site of mononucleotide polymorphic international number rs4412249 (of human 6th chromosome) 501st base in nucleotide sequence of SEQ ID NO: 3 present in a region including GMDS), base of the polymorphic region of the single-nucleotide polymorphism international number rs2763979 (SEQ ID NO: present in the region including HSPA1B of the human sixth chromosome) The 501st base in the nucleotide sequence of 4), and p in the gene region in which the polymorphic site exists A primer capable of amplifying a region comprising a base of at least one polymorphic site selected from the group consisting of bases of other polymorphic sites with a value (indicator of significant difference between normal tension glaucoma patients and healthy persons) of less than 0.05.
(b) the base of the polymorphic site of the single-nucleotide polymorphic international number rs3213787 (the 501st base in the nucleotide sequence of SEQ ID NO: 1 present in the region containing SRBD1 of the human second chromosome), and the single-nucleotide polymorphic international number rs735860 Base of polymorphic site (501st base in nucleotide sequence of SEQ ID NO: 2 present in a region containing ELOVL5 of human 6th chromosome), base of polymorphic site of mononucleotide polymorphic international number rs4412249 (of human 6th chromosome) 501st base in nucleotide sequence of SEQ ID NO: 3 present in a region including GMDS), base of the polymorphic region of the single-nucleotide polymorphism international number rs2763979 (SEQ ID NO: present in the region including HSPA1B of the human sixth chromosome) The 501st base in the nucleotide sequence of 4), and p in the gene region in which the polymorphic site exists Probe capable of hybridizing to a region containing bases of at least one polymorphic site selected from the group consisting of bases of other polymorphic sites with a value (indicator of significant difference between normal tension glaucoma patients and healthy persons) of less than 0.05 .
Within the gene region in which the polymorphic site of mononucleotide polymorphic international number rs3213787, the polymorphic site of mononucleotide polymorphic international number rs735860, the polymorphic site of mononucleotide polymorphic international number rs4412249, the polymorphic site of mononucleotide polymorphic international number rs2763979, and their polymorphic site Other polymorphic sites with p values (indicators of significant differences between normal tension glaucoma patients and healthy people) are below 0.05.
The present invention also provides a normal intraocular glaucoma test kit comprising the above reagent.
Primers and probes that are components of the reagents of the present invention may be oligonucleotides having a chain length of at least 15 nucleotides. When using this oligonucleotide as a primer, the length is 15 bp-100 bp normally, Preferably it is 17 bp-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 DNA which a primer can amplify is 15-1000 bp normally, Preferably it is 20-500 bp, More preferably, it is 20-200 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 DNA containing the polymorphic site. The length of DNA which can hybridize a probe is 16-500 bp normally, Preferably it is 20-200 bp, More preferably, it is 20-50 bp.
In the present invention, the primer capable of amplifying the region containing the polymorphic moiety may be one capable of initiating complementary chain synthesis toward the polymorphic moiety using DNA containing the polymorphic moiety as a template.
An arbitrary base sequence can be added to a primer in addition to the base sequence complementary to the base sequence of the area | region containing a polymorphic site. For example, in the primer for the polymorphic analysis method using the IIs type restriction enzyme, the primer which added the recognition sequence of the IIs type restriction enzyme is used. In addition, you may modify a primer. 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, a probe capable of hybridizing to a region containing a polymorphic moiety may be one capable of hybridizing with a polynucleotide having a nucleotide sequence of a region containing a polymorphic moiety, It is preferable to hybridize specifically to DNA having a nucleotide sequence. Here, "specifically hybridize" means under normal hybridization conditions, preferably under stringent hybridization conditions (e.g., Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, In the condition described in New York, USA, 2nd edition 1989), it is meant that cross hybridization with DNA other than DNA having a nucleotide sequence of a region including a polymorphic site is not significantly generated. More specifically, the probe which contains a polymorphic site in the base sequence of a probe is preferable. Alternatively, depending on the method of analyzing the base in the polymorphic site, it may be designed so that the terminal of the probe corresponds to the base adjacent to the polymorphic site. Therefore, although the polymorphic site is not contained in the base sequence of a probe itself, the probe containing the base sequence complementary to the area | region adjacent to a polymorphic site can also be shown as a preferable probe in this invention.
As the primer, modification of the nucleotide sequence, addition of the nucleotide sequence, or modification are allowed for the probe. For example, probes used in the Invader method have a sequence added 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 including 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 region of the present invention in the genome.
Those skilled in the art can design primers and probes according to an interpretation technique based on nucleotide sequence information for surrounding DNA regions including polymorphic sites. The base sequences constituting the primers and probes can be appropriately modified as well as the base sequences completely complementary to the base sequences of the genome.
Primers and probes can be synthesized by any method based on the nucleotide sequences constituting them. Based on a given nucleotide sequence, a technique for synthesizing an oligonucleotide having the nucleotide sequence is known. In the synthesis of oligonucleotides, arbitrary modifications may be introduced into the oligonucleotides using nucleotide derivatives modified with fluorescent dyes, biotin, or the like. Alternatively, a method of binding a fluorescent dye or the like to a synthesized oligonucleotide is also known.
The probe may be fixed in a solid phase (DNA array). The DNA array hybridizes sample DNA (or RNA) to multiple probes placed on the same plane, and by scanning the plane, hybridization for each probe is detected. DNA arrays are useful in that the reactions to many probes can be observed at the same time, for example to simultaneously interpret multiple polymorphic sites. As a method of fixing (arraying) nucleotides, an array based on oligonucleotides by Affymetrix Corporation can be exemplified. In an array of oligonucleotides, oligonucleotides are typically synthesized in situ. For example, in situ synthesis of oligonucleotides by lithography (Affymetrix), inkjet (Agilent), bead array (Illumina) and the like is known.
Oligonucleotides consist of a base sequence complementary to the region comprising the polymorphic site to be detected. The length of the nucleotide probe to be bonded to the substrate is usually 10 to 100 bp, preferably 10 to 50 bp, and more preferably 15 to 25 bp when the oligonucleotide is fixed.
Samples for SNP detection by the DNA array method can be prepared by methods well known to those skilled in the art based on biological samples collected from the subject. The biological sample is not particularly limited. For example, a DNA sample can be prepared from genomic DNA extracted from tissues or cells such as peripheral blood leukocytes, skin, oral mucosa, tears, saliva, urine, feces or hair of a subject. Using primers for amplifying the region containing the polymorphic site to be determined, a specific region of genomic DNA is amplified. At this time, a plurality of regions can be amplified simultaneously by the multiplex PCR method. The multiplex PCR method is a PCR method using a plurality of sets of primer sets in the same reaction solution. When analyzing a plurality of polymorphic sites, the multiplex PCR method is useful.
In general, in the DNA array method, amplification products are labeled while amplifying the DNA sample by the PCR method. As a label of the amplification product, a primer to which a label is issued is used. For example, first, genomic DNA is amplified by PCR using a primer set specific for a region including a polymorphic site. Next, biotin-labeled DNA is synthesized by a labeling PCR method using a biotin-labeled primer. The biotin-labeled DNA thus synthesized is hybridized to an oligonucleotide probe on a chip. The reaction liquid and reaction conditions of hybridization can be suitably adjusted according to conditions, such as the length of the nucleotide probe fixed to a solid phase, reaction temperature, etc. One skilled in the art can design the conditions of the appropriate hybridization. In order to detect hybridized DNA, avidin labeled with a fluorescent dye is added. The array is analyzed with a scanner, and fluorescence is used as an index to confirm the presence of hybridization.
When an example of the procedure which performs the test method of this invention using the DNA array method is shown, after preparing the solid phase to which the DNA and nucleotide probe containing the polymorphic site prepared from the subject were fixed, the DNA and the solid phase are contacted. Subsequently, the base species of the polymorphic site is determined by detecting DNA hybridized to a nucleotide probe immobilized on a solid phase.
In this specification, "solid phase" means the material which can fix | immobilize a nucleotide. The solid phase is not particularly limited as long as it is possible to fix nucleotides. Specific examples thereof include a solid phase including a microplate well, 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 this specification, a "substrate" means the plate-shaped material which can fix | immobilize a nucleotide. In the present invention, nucleotides include oligonucleotides and polynucleotides.
In addition to the above method, in order to detect the base of a specific site, the allele specific oligonucleotide (ASO) hybridization method can be used. The allel specific oligonucleotide (ASO) consists of a nucleotide sequence that hybridizes to the region where the polymorphic site to be detected exists. When ASO is hybridized to the sample DNA, if a mismatch occurs in the polymorphic region depending on the polymorphism, hybridization efficiency is lowered. Mismatch can be detected by a Southern blot method or a method using the property of quenching by intercalating a special fluorescent reagent in the gap of the hybrid. Moreover, mismatch can also be detected by the ribonuclease A mismatch cleavage method.
The reagents and kits of the present invention may include various enzymes, enzyme substrates, buffers, and the like, depending on the method of identifying the base. As an enzyme, the enzyme required for the various analysis methods illustrated as said base identification method, such as DNA polymerase, DNA ligase, or IIs restriction enzyme, can be shown. As the buffer, a buffer suitable for maintaining the activity of the enzyme used in these interpretations is appropriately selected. As the enzyme substrate, for example, a substrate for complementary chain synthesis or the like is used.
In addition, the reagent and kit of the present invention can be attached to a control having a clear base at the polymorphic site. As a control, genomic DNA or fragments of genomic DNA having a clear base species of a polymorphic site can be used. Genomic DNA may be attached as a control that is extracted from a cell, or a cell or a fraction of cells may be attached as a control, and the user may extract genomic DNA therefrom. When cells are used as a control, it can be proved that the extraction operation of genomic DNA was performed correctly according to the result of the control. Alternatively, DNA consisting of a nucleotide sequence containing a polymorphic site may be used as a control. Specifically, you may use as a control the YAC vector and BAC vector containing DNA from genomic origin from which the base species in a polymorphic site became clear. Alternatively, a vector obtained by cutting out and inserting only tens to hundreds of bp corresponding to a polymorphic region may be used as a control.
Example
EMBODIMENT OF THE INVENTION 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 human specimens were collected under the permission of the University of Yokohama Medical University, Ethics Committee, and collected NTG patient blood leukocytes that satisfy the strict criterion of age, intraocular pressure, refractive error, and the like.
NTG's diagnostic criteria were based on the criteria of the Japanese Glaucoma Society.
As primary, 305 patients and 355 healthy persons were narrowed down to 53 gene regions. As continuous replication, 4 SNPs with high expected efficacy were identified in 214 different patients and 257 healthy individuals. The four genes are shown in the table, and the values calculated for the entire sample (overall) in addition to the results of primary and replication respectively.
Genotyping was performed using a GeneChip Human Mapping 500K Array Set (Affymetrix) according to a standard protocol recommended by Affymetrix.
The interpretation of GWAS, including statistical interpretation, was Helixtree SVS 7 (Golden Herix. Inc. Bozeman, Montana, USA), and Haploview v4.1.
Haploview v4.1 was also used to estimate the LD structure of 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 it was demonstrated that it is an effective SNP for diagnosing NTG.
Figure pct00021
New results of increasing the number of patients and healthy people (adding data from 46 patients and 46 healthy people) to the data are shown in the table below. The same propensity for allergy was seen in additional patients and healthy individuals.
Figure pct00022
All publications, patents and patent applications cited herein are hereby incorporated by reference.
Industrial availability
The invention is applicable to life sciences, medicine, ophthalmology and diagnostics.
Array-free text
<Array number 1>
SEQ ID NO: 1 shows a nucleotide sequence of 1000 base length comprising the polymorphic site of rs3213787 at the 501st (r = A / G).
<Array number 2>
SEQ ID NO: 2 shows a nucleotide sequence of 1000 base length comprising the polymorphic site of rs735860 as the 501st (y = C / T).
<Array number 3>
SEQ ID NO: 3 shows a nucleotide sequence of 1000 base length comprising the polymorphic site of rs4412249 at the 501st (r = A / G).
<Array number 4>
SEQ ID NO: 4 shows a nucleotide sequence of 1000 base length comprising the polymorphic region of rs2763979 at 501st (y = C / T).
<110> Menicon Co., Ltd. <120> Normal tension glaucoma-susceptibility gene and use <130> FP-152PCT <150> JP P2010-120758 <151> 2010-05-26 <160> 4 <170> PatentIn version 3.1 <210> 1 <211> 1001 <212> DNA <213> Homo sapiens <400> 1 tttctgctat acctgtttca gtgaaaacgg tccagatcag ggaatttcat gctgcattag 60 cagacttgtg taataaatgc aaaactgtga acaaaaaagc agctcttcta aaaccatggg 120 ggaaaagaag attatataac atgcgattat agtatgataa aaattttgac aggtttcttg 180 tggaatcagg gtaaaaatat ggtaaccata ccttgaaccc tcagaattta tcttttggtt 240 actgccaggt ttgacagatg catgcaagag gcttaactag tgaaaaatct ctactctcac 300 gtggtgggaa acagctcttt tccatgttaa ccttatttaa attctattag caacactaat 360 acactcaatg gttaccatat gctcttcact ttagacctgc aatattaaaa atgataatgt 420 gaattgttat aattcttttc ttaaatgatg tcttcccctc tgattactta aagcaatgta 480 taaacccata gacgttctct rcttgacaac tttatacacg tgttcccttc ttctagccga 540 gtttggactg gacagccagt taaactcaag attctgtgat cttcagccac atatttgtct 600 caccttaaca aaacttctga acagatgtta atatccactc ccacaaagct gacacattct 660 tctacaacac tgtccagtgt tgccttgagt aaagtctggg atacgtcatg ctgaaaagac 720 aaagatcaaa tattaacagt gaaggagatg tagttcttgg actattccat tactctgcct 780 atattactgg tggttagcta acacacacca tggtaaattt cttcattcct cttagacaca 840 ctgttagcag aattaagcta aagtcaaggc atctgtggct tcttactctt ttaagctatg 900 catatatacc taatttgaat ataacagtac cagtattttt cccttgcctg aaattgtgcc 960 cagagcagtg gttctcaaac ttaagcttgc attaaaatca c 1001 <210> 2 <211> 1001 <212> DNA <213> Homo sapiens <400> 2 tctcatgttc attcattcac aaattcattt attctaatat ttgagactct cctatgtggc 60 aggcaaaaga aaaagccctt gatttcaata tagatacaac acaatggaaa tattacagag 120 aaaaataaag ccaagtcaaa gagaggtgtg tgtggggagg gatgtgagga atgatatttt 180 agatctggta gttggggagg cctctctggg aaggtggcat ttaagtgcag ggacaagcca 240 tgcaaataaa catctggagg tgagaacaaa ggccttgaga tggaagtggc tcctctagag 300 aaccaggcaa gaggtgaggg cagctgaatg tggtgagcaa ggaagaggat gggacatgtg 360 agaggggtca gggccagctc caggagcctg taggccatgg aaaggcatag gattttattc 420 tccgtgaagc aagtgcctag aggtggctcc cagacacagg caagctagtg acctcaaggg 480 ggccttggtc ctgctccatc ygaagagctg attcattaga cttttgcagt ccctgatccc 540 aggagctaca accttagatt gccagggaac tggaagcatt taattaggga ttctggggag 600 aagctgtggg tcctgtgata aggtccctgt ggctgcaaga cccaccctcc tggaggtttc 660 atgattcaga tgagctgttc tggcactctg ggtccactag aagctgatgg gatttttaac 720 agaatgttat tcccactgtg ccaccagcag gtttttatgg cctcatgtga ccattaactg 780 ttgcgtgggg atcataattc taagagaccc ccctggagac catatggtct gtatctattt 840 tcttgaatcc tagaaccatg ctgaggttta ggaggttagg taacttaaca agggcacatg 900 gttattgagt gatggcagga tttcatctct gatctaattg gctggtgata tggtttggct 960 ctgtgttccc accagaatct catcttgaat tgtaatcccc a 1001 <210> 3 <211> 1001 <212> DNA <213> Homo sapiens <400> 3 ttgcccactt tttaattggg ttgtcttttt attactgagt tgtaagagtt tttagatatt 60 ctaaatctct atcccttatc agatacatga tttgcaaata gtttctctca ttctgtgggt 120 tgccattctc tttcttcatg gtatcttttg aaacacaaag gtttctaatt tttttctttt 180 aagagtttta tgcttttagc tcttacatta ggtttttgaa actgatgcac tgattttaaa 240 aaccatatga aatagcaaag gatccagaat aaccagaaaa agaacaaagt tggaggactc 300 acacttccta aattccaaac ttataataca agcaacagta ataaagaaag tgctgtactg 360 gcataagaat agatgcacaa atcaatggac tagaaacatg agtccagaaa aaaacccatg 420 tgtctaggat cgactgattt cgacaagagt tgctaggacc attcagtggg aaagagtagt 480 cttttcaaca aatggtgcac rgacaactgg agagctacat gtaaaagaat gaagtttgcc 540 cttacttcat agcatatata aaacttaact caaatggatc taagacctaa atgtagcagt 600 ctcagtttta tatttaaata cattcactat ttgccaccaa ttccttttac tgtggcttta 660 ctattccaat tttattttgc ttcatcaatt tgctggctga attatactct cagatggttt 720 ccaagtagaa gtgcataggt gctgaataat cacaattgtt taatattcgt ttttaaaagt 780 ttgttttaaa atggtcttga agggaaaatt ggatttatat aaagttctgg aatcacattt 840 cctttgttcc accatcttct aagaccaaga acttattcat tcatttattc atttaacaaa 900 cattgttgcg tgcctaccgc atgtcagacg gtagtggcga aagacagtgt ctctgccctc 960 agggctcctt tatcctattg gaaggaaata atcaacaaaa g 1001 <210> 4 <211> 1001 <212> DNA <213> Homo sapiens <400> 4 gagttcgaga ccagcctgac caacatgggg aaaccctgtc tctactaaaa atacaaaaat 60 tagccaggcg tggtggcgca cgcctgtaat cctagctact caggaggctg aggtaggaga 120 attgcttaaa cccgggaggc ggaggttgca gtgagccgag atcacgccac tgcactccag 180 cttgggcgac agagcgagac tgtctcaaaa cgaaaacaac aaacttaaga cacataacct 240 gaggtgttaa gaggagctag taactagaac ctgggtccca acccctcctg ctttccagca 300 tcactccaca cagtttgctt aaagagggcc acctgccaaa cagctgtagt atgtgatgtt 360 aaagagagct aaacaccccc cgcacctccc tcccagggtc accatcttgt taaatttgac 420 ctaaaaacgg taacagccta ggggtttcag ggacagacag aaaatcttac tcgggactgt 480 gaggtcctac ttctacacac ygtccaggag tgaaccagga attgagaaag taggaaggag 540 gtgtcccaga ccccaagcta ggaatgggga gggaaatgga ggaatcccaa atgccttaag 600 gacggcctac atactaagga aaattttttt ctaactcctg gttgcagctg aggggagcgg 660 ctgagggcgg ggacaggggt gcggcggacc cactgctccc attacccgac cagcgcctcc 720 cttcctcctt ggatgggtgc ccctgtcttg ctaagaactg cctgtttaca caactgcttt 780 ccttgtgaaa atttaaaggc tcctattccc agttgttcta tccttgtagg ttaaagatta 840 tgtcaaaaac tatattgcat tatctctttc cttctccttc ccattaagac ggaaaaaaca 900 tccgggagag ccggtccgtt tctcaggcag actaggccat taggtgcctc ggagaaagga 960 cccaaggctg ctccgtcctt cacagacaca gtccaatcag a 1001

Claims (6)

  1. Base of the polymorphic site of mononucleotide polymorphic international number rs3213787 (the 501st base in the nucleotide sequence of SEQ ID NO: 1 present in a region containing SRBD1 of the human second chromosome), and of the polymorphic site of mononucleotide polymorphic international number rs735860 Base (501st base in nucleotide sequence of SEQ ID NO: 2 present in a region containing ELOVL5 of the human sixth chromosome), base of the polymorphic site of the single-nucleotide polymorphism international number rs4412249 (GMDS of the human sixth chromosome) 501st base in nucleotide sequence of SEQ ID NO: 3 present in a region thereof, and nucleotide of SEQ ID NO: 4 present in a region including HSPA1B of the sixth chromosome of the polymorphic region of the single-nucleotide polymorphism international number rs2763979. The 501st base in the sequence), and the p value (in the gene region where those polymorphic sites exist) The tension glaucoma NTG inspection method, comprising a patient and a healthy person identification of at least one nucleotide of the polymorphic site that is significantly different indices) selected from the group consisting of the nucleotide of the polymorphic site of the other is less than 0.05 between.
  2. The method of claim 1,
    Other polymorphisms with a p-value (indicative of significant differences between patients with normal-tension glaucoma and healthy persons) within the gene region in which the polymorphic sites of the rs3213787, rs735860, rs4412249 and rs2763979 exist, are among the other polymorphic polymorphic polymorphisms. Method that is polymorph in polymorphism and chain imbalance of number rs3213787, rs735860, rs4412249 or rs2763979.
  3. 3. The method of claim 2,
    A polymorph in polymorphic and chain unbalance of a single base polymorphic international number rs3213787, rs735860, rs4412249 or rs2763979 is a polymorph in the LD block of a single base polymorphic international number rs3213787, rs735860, rs4412249 or rs2763979.
  4. A reagent for performing a normal intraocular glaucoma test, comprising at least one component selected from the group consisting of the following components (a) and (b).
    (a) 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 the human second chromosome), and the single-nucleotide polymorphism international number rs735860 of Base of polymorphic site (501st base in nucleotide sequence of SEQ ID NO: 2 present in a region containing ELOVL5 of human 6th chromosome), base of polymorphic site of mononucleotide polymorphic international number rs4412249 (of human 6th chromosome) 501st base in nucleotide sequence of SEQ ID NO: 3 present in a region including GMDS), base of the polymorphic region of the single-nucleotide polymorphism international number rs2763979 (SEQ ID NO: present in the region including HSPA1B of the human sixth chromosome) The 501st base in the nucleotide sequence of 4), and p in the gene region in which the polymorphic site exists A primer capable of amplifying a region comprising a base of at least one polymorphic site selected from the group consisting of bases of other polymorphic sites with a value (indicator of significant difference between normal tension glaucoma patients and healthy persons) of less than 0.05.
    (b) the base of the polymorphic site of the single-nucleotide polymorphic international number rs3213787 (the 501st base in the nucleotide sequence of SEQ ID NO: 1 present in the region containing SRBD1 of the human second chromosome), and the single-nucleotide polymorphic international number rs735860 Base of polymorphic site (501st base in nucleotide sequence of SEQ ID NO: 2 present in a region containing ELOVL5 of human 6th chromosome), base of polymorphic site of mononucleotide polymorphic international number rs4412249 (of human 6th chromosome) 501st base in nucleotide sequence of SEQ ID NO: 3 present in a region including GMDS), base of the polymorphic region of the single-nucleotide polymorphism international number rs2763979 (SEQ ID NO: present in the region including HSPA1B of the human sixth chromosome) The 501st base in the nucleotide sequence of 4), and p in the gene region in which the polymorphic site exists Probe capable of hybridizing to a region containing bases of at least one polymorphic site selected from the group consisting of bases of other polymorphic sites with a value (indicator of significant difference between normal tension glaucoma patients and healthy persons) of less than 0.05 .
  5. The method of claim 4, wherein
    Reagent in which the probe is fixed in the solid phase.
  6. A normal-tension glaucoma test kit comprising the reagents of claim 4 or 5.
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