JP2006325528A - Diagnosis and prophylaxis of diabetic retinopathy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for judging (diagnosing) the crisis of diabetic retinopathy at the early stage of a crisis or its crisis possibility, a kit for judging (diagnosing) the same and a method for effectively preventing the crisis of a diabetic retinopathy and to obtain a prophylactic effectively preventing the crisis of a diabetic retinopathy. <P>SOLUTION: The method for simply judging the crisis of diabetic retinopathy or its crisis possibility comprises detecting presence of variation in Ang-1 (Angiopoietin-1) gene or in a protein being its translation product since there is a causal relationship between the crisis of diabetic retinopathy and the variation. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to the diagnosis and prevention of diabetic retinopathy, and more particularly to the diagnosis and prevention of diabetic retinopathy using angiopoietin-1 gene or a protein that is a translation product thereof.

  Diabetic retinopathy (hereinafter also referred to as “DR”) is a major chronic disease that is a major complication of diabetic nephropathy and diabetes along with diabetic neuropathy.

  The blood of a diabetic patient contains a lot of sugar and has high viscosity, and therefore clogs the capillaries of the retina and places a burden on the blood vessel wall, resulting in a lack of oxygen and nutrients necessary for the retina. DR is a disease that occurs as a result, and exhibits symptoms such as fundus bleeding and vitreous hemorrhage. Diabetic retinopathy can be divided into three stages according to the progression process: simple DR, preproliferative DR, and proliferative DR.

  Simple DR is an early stage of DR. At this stage, capillary aneurysms and spotted bleeding are seen in the capillaries that send oxygen to the retina. In addition, proteins and the like may accumulate in the retina and cause hard vitiligo.

  The pre-proliferation type DR is a stage where the simple type DR has progressed. At this stage, there are symptoms that the number of vitiligo increases and the capillaries are partially blocked.

  Proliferative DR is a stage where pre-proliferative DR has further progressed. Capillaries in the retina are blocked and oxygen sent to the retina is insufficient, so new neovascularization appears to compensate for this. However, since the new blood vessels are fragile, they tend to bleed, and if the retinal detachment occurs or further progresses, it can lead to blindness.

  In the case of DR, in the case of simple DR, if there is no bleeding or edema in the center of the retina, there is no noticeable subjective symptom, and when the subjective symptom appears, it is already in a state of blindness (proliferative DR state) There is almost always. DR is also the leading cause of blindness in adults in Japan. Therefore, the DR patient suffers great physical and mental pain throughout his life.

  The diagnosis of DR is mainly performed by examining the fundus. Specifically, (1) “fundus examination” in which the inside of the eyeball is observed from the pupil through the lens by shining light on the eyeball, and (2) while injecting the dye from the vein This is performed by “fluorescent fundus imaging” or the like for examining the fundus.

  In addition, in the treatment of DR, the treatment means to be selected usually differs depending on the progression of the pathological condition. For example, in the case of the above-mentioned simple DR, a therapeutic method for controlling blood glucose medically, including administration of a vascular enhancing agent and antiplatelet therapy, and a therapeutic method for suppressing retinal changes with a drug are employed. On the other hand, at the stage of pre-proliferation type DR or proliferative type DR, “retinal photocoagulation therapy” is adopted in which the retina is burned with the heat of a laser beam to suppress the formation of blood vessels. Further, when symptoms of vitreous hemorrhage or retinal detachment are observed at the stage of proliferative DR, vitreous surgery may be performed. However, vitrectomy often causes complications and the post-operative course may be poor.

  By the way, the onset mechanism of DR is gradually being clarified by recent studies. Specifically, it is clear that DR develops when leukocytes first adhere to the retinal vasculature, followed by damage to vascular endothelial cells and destruction of the blood-retinal barrier. It was made. In an experiment using diabetic rats, the usefulness of angiopoietin-1 (hereinafter also referred to as “Ang-1”) was reported as a factor that inhibits this process (Non-patent Document 1). See).

  The main actions of Ang-1 include the lining of new blood vessels induced by vascular endotherial growth factor (hereinafter also referred to as “VEGF”), the action of promoting the formation of new blood vessels, and perivascular smooth muscle It has the effect of promoting the creation of Ang-1 acts on Tie-2 (tyrosine kinase with immunoglobulin and endothelial growth factor homology domains) receptor, which is an angiogenesis-related tyrosine kinase type receptor on the vascular endothelium, and adheres to fibronectin via integrin. Facilitate. In addition, it has been clarified that Ang-1 enhances the survival effect by promoting migration of vascular endothelial cells and simultaneously suppressing cell death of vascular endothelial cells during this series of intracellular signal transduction. (See Non-Patent Document 2).

  On the other hand, the present inventors have revealed that a mutation in the Ang-1 gene has a causal relationship with the onset of rheumatoid arthritis (hereinafter also referred to as “RA”) (see Patent Document 1). ). Patent Document 1 shows that the following mutations in the Ang-1 gene are significantly higher in the RA patient population.

The mutation in the Ang-1 gene is “GGT” between the 804th base and the 805th base of the base sequence (accession number: AB084454) registered in GenBank as the cDNA of the Ang-1 gene. Mutation (base sequence registered in GenBank (accession number: U83505)) in which 3 bases encoding glycine were inserted. The position of the mutation in the Ang-1 gene was present between the coiled-coil domain and the fibrinogen-like domain present in the Ang-1 molecule.
JP 2003-204790 A (published July 22, 2003) Antonia M. Joussen et al., Suppression of diabetic retinopathy with Angiopoetin-1, American journal of pathology, 160, 1683-1693 (2003) Christopher D. Kontos et al., Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt, Molecular and Cellular Biology, 18, 4131-4140 (1998)

  As described above, DR is a disease that finally causes serious symptoms, but early detection is difficult because there is no subjective symptom at the initial stage of onset. In addition, the diagnosis of DR is mainly performed by a subjective diagnosis method called a fundus examination. Therefore, at present, establishment of a technique that can objectively and accurately diagnose the onset of DR or a technique that can diagnose the possibility of developing DR in the future is strongly desired.

  As a method for objectively diagnosing the onset of DR or the possibility of the onset in the future, a diagnostic method using a molecular biological technique is considered to be one of the effective diagnostic methods. However, as shown in Non-Patent Document 1, although there are reports of genes that are causally related to the onset of DR, gene abnormalities (mutations) that have been revealed to have a causal relationship to the onset of DR have never been found so far. unknown. Therefore, it has not yet established a technique for early diagnosis of the onset of DR using molecular biological techniques or the possibility of developing in the future.

  The present invention has been made in view of the above problems, and its object is to determine (diagnose) the onset or possibility of onset of DR early in the onset, its determination (diagnostic) kit, and DR onset It is an object to provide an effective preventive method and preventive agent for the disease.

  As a result of intensive studies in view of the above problems, the present inventors have found a mutation of a gene that is causally related to the onset of DR, and have completed the present invention.

  That is, the method for determining the onset of DR or the possibility of its onset according to the present invention is a method for determining the onset of diabetic retinopathy or the possibility of its onset using a sample isolated from a living body, A step of detecting the presence or absence of a mutation in a polynucleotide encoding an angiopoietin-1 protein (hereinafter also referred to as “Ang-1”), and the mutation is a mutant angiopoietin-1 protein (hereinafter referred to as “angiopoietin-1 protein”). It is characterized by being a 3-base deletion mutation encoding glycine, which is the 269th amino acid of "mutant Ang-1".

  The method for determining the onset of DR or the possibility of the onset according to the present invention is a method for determining the onset of the diabetic retinopathy or the possibility of the onset using a sample separated from a living body, A step of detecting the presence or absence of a mutation in the angiopoietin-1 gene (hereinafter also referred to as “Ang-1 gene”), and the mutation is a mutant angiopoietin-1 gene (hereinafter referred to as “mutant Ang”). -1 gene "), a 3 base deletion mutation of" GGT "(G and T represent guanine and thymine, respectively) which is the 805th to 807th base in the translation region Yes.

  Further, the method for determining the onset of DR or the possibility of the onset according to the present invention is a method for determining the onset of diabetic retinopathy or the possibility of the onset using a sample separated from a living body, A step of detecting the presence or absence of a mutation in Ang-1, and the mutation is a deletion mutation of glycine, which is the 269th amino acid of mutant Ang-1.

  The kit for determining the onset of DR or the possibility of its onset according to the present invention is characterized by using any of the above-described methods for determining the onset of DR or the possibility of its onset.

  The kit for determining the onset of DR or the possibility of the onset thereof preferably includes at least one of a primer, a probe, and an antibody.

  The primer is preferably a primer for amplifying a region containing the 805th to 807th bases in the translated region of the mutant Ang-1 gene.

  The probe preferably binds to a region containing the 805th to 807th bases in the translation region of the mutant Ang-1 gene.

  The antibody may be the above-mentioned mutant Ang-1, or a normal angiopoietin-1 protein lacking glycine, which is the 269th amino acid of the mutant Ang-1 (hereinafter referred to as “normal Ang-1”). It is preferable to recognize only one of the above.

  The method for preventing the onset of DR according to the present invention is directed to a mutant Ang-1 having glycine as the 269th amino acid in a person having a normal Ang-1 lacking the 269th glycine of the mutant Ang-1. Alternatively, it is characterized by complementing a low molecular weight compound as an agonist of a receptor protein having a ligand encoding the mutant Ang-1 or the mutant Ang-1 as a ligand.

  The preventive agent for the onset of DR according to the present invention is a preventive agent used for preventing the onset of diabetic retinopathy, and is a normal type lacking the 269th glycine of the mutant angiopoietin-1 protein. A person having an angiopoietin-1 protein to be administered, a mutant angiopoietin-1 protein having glycine as the 269th amino acid, a DNA encoding the mutant angiopoietin-1 protein, or the mutation Type angiopoietin-1 protein is mainly composed of a low molecular weight compound as an agonist of a receptor protein that serves as a ligand.

  As described above, according to the present invention, by using the Ang-1 gene or a protein that is a translation product thereof to detect the presence or absence of mutations in them, the onset of DR or the possibility of the onset is determined. be able to. Therefore, it is possible to easily determine the onset of DR or the possibility of the onset thereof. Furthermore, by using the mutant Ang-1 gene or its translation product, mutant Ang-1, it is possible to apply the method to the prevention of DR and the development of preventive drugs.

  Prior to completing the present invention, the present inventors have found that the Ang-1 gene mutation disclosed in Patent Document 1 may affect signal transduction via the Tie-2 receptor. Derived independently. More specifically, we believe that Ang-1 gene mutations can have a significant impact on the stabilization of the retinal vasculature and the mature organization of blood vessels that underlie the pathogenesis of DR. I derived something. Therefore, the present inventors examined whether the Ang-1 gene mutation is a factor associated with the onset of DR.

  As a result, the present inventors have a gene having a base sequence (accession number: U83505) registered in GenBank as an Ang-1 gene cDNA, that is, a gene into which three bases of “GGT” are inserted. Revealed that there were significantly fewer individuals in DR patients. That is, the present inventors have found that the insertion mutation of “GGT” in the Ang-1 gene may be a suppression regulator of the onset of DR. Furthermore, the above-mentioned “GGT” 3 base sequences that do not have 3 bases and are often found in healthy individuals were registered in Genbank as accession number: AB0844454. Thus, the present inventors have found that the Ang-1 gene having the above-mentioned “GGT” 3-base insertion mutation is a regulator for controlling the onset of DR, and completed the present invention. It was.

  An embodiment of the present invention will be described as follows, but the present invention is not limited to this.

  In the present specification, unless otherwise specified, A, C, G and T represent adenine, cytosine, guanine and thymine bases. For amino acids and amino acid residues, the one-letter code or three-letter code defined by IUPAC and IUB is used.

  In the present specification, the term “gene” means at least a polynucleotide such as genomic DNA, cDNA, mRNA and the like, and the gene according to the present invention includes the above-mentioned cDNA of the mutant Ang-1 gene, Examples include mRNA having a base sequence corresponding to the base sequence of this cDNA, genomic DNA serving as a template for this mRNA, and the like. The “gene” includes not only double-stranded DNA but also single-stranded DNA and RNA such as sense strand and antisense strand constituting the DNA. Furthermore, the “gene” may include a sequence of an untranslated region (UTR), a promoter sequence, a vector sequence (including an expression vector sequence) and the like in addition to the translation region.

<1. Angiopoietin-1 gene and angiopoietin-1 protein>
In this specification, “Ang-1 gene” means a nucleotide sequence (accession number: AB084544) registered in GenBank, a nucleotide sequence (accession number: U83508), and the like on a nucleotide sequence database. It is a general term for genes having a nucleotide sequence disclosed as an angiopoietin-1 gene.

  On the other hand, in the present specification, the “mutant Ang-1 gene” is a gene encoding mutant Ang-1 described later. Specifically, it is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and having glycine as the 269th amino acid in the sequence. For example, a gene having the base sequence shown in SEQ ID NO: 2 and having 3 bases “GGT” as the 805th to 807th bases in the sequence can be mentioned as an example. The base sequence shown in SEQ ID NO: 2 corresponds to the base sequence (accession number: U83508) registered in GenBank as the Ang-1 gene cDNA. The 805th to 807th bases in SEQ ID NO: 2 correspond to the 1114th to 1116th bases of the base sequence (accession number: U83508) registered in GenBank as cDNA for the Ang-1 gene. To do.

  Further, in the present specification, the “normal Ang-1 gene” is a gene encoding normal Ang-1 described later, for example, a base sequence registered in GenBank (accession number: AB0844454) For example, a gene having

  Furthermore, the normal Ang-1 gene and the mutant Ang-1 gene according to the present invention include the base sequence registered in GenBank (accession number: AB084454) and the base shown in SEQ ID NO: 2, respectively. A polynucleotide in which a mutation such as insertion, substitution, and / or deletion is inserted into a part of the sequence, and encodes a normal Ang-1 and a protein having a function equivalent to that of the mutant Ang-1 It goes without saying that the genes to be included are included. Note that the number of mutations may be one or more. Furthermore, a polynucleotide having an additional sequence 5 'upstream and / or 3' downstream of the base sequence shown in SEQ ID NO: 2 is also included in the mutant Ang-1 gene according to the present invention.

  In the present specification, “Ang-1” is a general term for the translation product of the Ang-1 gene and a protein whose amino acid sequence is disclosed as angiopoietin-1 on various databases.

  On the other hand, in this specification, “mutant Ang-1” refers to a translation product of the above-mentioned mutant Ang-1 gene. For example, a protein having the amino acid sequence shown in SEQ ID NO: 1 and having glycine as the 269th amino acid in the sequence can be mentioned.

  In the present specification, “normal Ang-1” refers to the translation product of the normal Ang-1 gene. For example, a protein lacking the 269th glycine in the amino acid sequence shown in SEQ ID NO: 1 can be mentioned.

  The normal type Ang-1 and the mutant type Ang-1 may further contain an additional polypeptide. Examples of the case where the additional polypeptide is added include a case where the protein is epitope-tagged with a His tag or the like. Furthermore, a protein in which a mutation such as insertion, substitution, and / or deletion is inserted into a part of the amino acid sequence shown in SEQ ID NO: 1, and has a function equivalent to that of mutant Ang-1 However, it goes without saying that it is included in the mutant Ang-1 according to the present invention. Further, the same can be said for the normal type Ang-1. Note that the number of mutations may be one or more.

<2. Method for determining onset of DR or possibility of onset>
According to the present invention, a method for determining the onset of DR or its likelihood of occurrence (in other words, a DR diagnosis method) is performed by determining whether Ang-1, a polynucleotide encoding Ang-1, or the presence of a mutation in the Ang-1 gene. What is necessary is just to include the process to detect, and it does not specifically limit about another structure.

  The mutation of Ang-1 is preferably a deletion mutation of glycine, which is the 269th amino acid in the mutant Ang-1. For example, a deletion mutation of glycine that is the 269th amino acid in the amino acid sequence shown in SEQ ID NO: 1 can be mentioned.

  Further, the mutation of the polynucleotide encoding Ang-1 is preferably a 3-base deletion mutation encoding glycine, which is the 269th amino acid of the mutant Ang-1. For example, a 3 base deletion mutation of “GGT” encoding glycine, which is the 269th amino acid in the amino acid sequence shown in SEQ ID NO: 1 can be mentioned.

  Furthermore, the mutation of the Ang-1 gene is preferably a 3 base deletion mutation of “GGT” which is the 805th to 807th base in the translation region of the mutant Ang-1 gene. For example, a 3 base deletion mutation of “GGT” which is the 805th to 807th base in the base sequence shown in SEQ ID NO: 2 can be mentioned.

  A specific configuration of the method for determining the onset of DR or the possibility of the onset according to the present invention will be described in detail below. Specifically, as a method for determining the onset or possibility of onset of DR according to the present invention, (A) a method using genomic DNA, (B) a method using mRNA (cDNA), (C) a protein according to the present invention Can be mentioned. Hereinafter, these three methods will be described.

(A) Method Using Genomic DNA This determination method using genomic DNA can be performed, for example, as follows.

  The genome of the subject can be obtained from all cells of the human body by a conventional method, but can be obtained from, for example, hair, organs, peripheral lymphocytes, synoviocytes and the like. Further, the obtained cells can be obtained by culturing and proliferating. It can also be obtained from peripheral blood.

  The obtained genome can be obtained by, for example, a commonly performed gene amplification method such as PCR (Polymerase Chain Reaction) method, NASBA (Nucleic acid sequence based amplification) method, TMA (Transcription-mediated amplification) method and SDA (Strand Displacement Amplification) method. Can be amplified and used.

  The method for detecting the presence or absence of a mutation on the genome is not particularly limited. For example, an allyl-specific oligonucleotide probe method, an oligonucleotide ligation assay method, a PCR-SSCP method, a PCR- Examples thereof include a CFLP method, a PCR-DRFA method, an invader method, an RCA (Rolling Circle Amplification) method, and a primer oligo base extension (Primer Oligo Base Extension) method.

  For example, after amplification of the region containing the mutation position on the Ang-1 gene from the genome, the resulting PCR product can be subcloned and directly sequenced to detect the presence or absence of the mutation on the genome.

  Moreover, the presence or absence of a mutation on the genome can be detected by using a region containing the mutation position on the Ang-1 gene as an oligonucleotide probe. In this case, for example, a DNA chip is constructed by fixing an oligonucleotide consisting of the base sequence of the region including the mutation position on the chip, and the DNA chip is used for detecting the presence or absence of the mutation. The case where it uses is mentioned. In this case, the length of the oligonucleotide probe is preferably 7 to 50 nucleotides or 10 to 30 nucleotides including the mutation position, and more preferably 15 to 25 nucleotides.

  The presence or absence of mutations in the genome can also be detected by detecting differences in the size of genomic fragments to be cleaved by Southern blotting or the like using an appropriate restriction enzyme.

  Thus, by detecting a mutation on the genome, it is possible to objectively and easily perform diagnosis (determination of onset or possibility of onset) of a subject's DR. Specifically, when a normal Ang-1 gene (3-base deletion Ang-1 gene) is detected in a homozygous state, it is determined that the possibility of developing DR or the possibility of developing in the future is high. be able to. On the other hand, when the mutant Ang-1 gene (3-base insertion Ang-1 gene) and the normal Ang-1 gene are detected in a heterogeneous state or the mutant Ang-1 gene is detected in a homozygous state, It can be determined that the possibility of developing DR or the possibility of developing it in the future is low.

  In addition, the primer and probe used by the said determination method can be produced with a DNA synthesizer etc. by a conventional method.

(B) Method using mRNA (cDNA) When using mRNA, for example, cDNA is prepared from mRNA extracted from the subject's cells by reverse transcription, and after amplification of the region containing the mutation position, amplification is performed as described above. The presence or absence of the mutation can be detected by directly sequencing the fragment base sequence, using a DNA chip, or using the RFLP (Restriction Fragment Length Polymorphism) method.

The primer used for amplification of the region including the mutation position is not particularly limited. For example, an amplification reaction using cDNA as a template is possible by a combination of the following primers.
Sense primer 1: 5'-CCACCAACAACAGTGTCCTT-3 '(SEQ ID NO: 3)
Sense primer 2: 5'-CAACCTTGTCAATCTTTGC-3 '(SEQ ID NO: 4)
Sense primer 3: 5'-GCTGGCAGTACAATGACAG-3 '(SEQ ID NO: 5)
Antisense primer 1: 5'-TCAAAAATCTAAAGGTCGAAT-3 (SEQ ID NO: 6)
Antisense primer 2: 5'-CAGCTTGATATACATCTGCACAG-3 '(SEQ ID NO: 7)
In this way, by detecting a mutation in mRNA (cDNA), it is possible to easily perform diagnosis (determination of onset or possibility of onset) of a subject in the same manner as in the method using genomic DNA. it can.

(C) Method Using Protein When using a protein prepared from a subject's cells, a method of detecting the presence or absence of glycine insertion at the mutation position in the amino acid sequence of SEQ ID NO: 1 can be mentioned. The detection of the presence or absence of this glycine insertion may be in accordance with a normal protein sequencing method. For example, only glycine insertion type protein (mutant type Ang-1) or glycine deficient type protein (normal type Ang-1) is used. A method for preparing an antibody to be recognized and detecting it by ELISA, a method for isolating a protein, directly or if necessary cleaving with an enzyme, etc., and detecting a mutation using a protein sequencer, a mutation in the isoelectric point of an amino acid And a method of detecting a difference in mass by mass spectrometry. Preferably, an antibody that recognizes only normal Ang-1 or mutant Ang-1 is prepared and detected by ELISA The method of doing is mentioned.

  Thus, by detecting the presence or absence of glycine insertion at the mutation position, it is possible to objectively and easily perform diagnosis (determination of onset or possibility of onset) of a subject's DR. Specifically, when mutant Ang-1 is not detected (in other words, when only normal Ang-1 is detected), the possibility of developing DR or the possibility of developing in the future is It can be determined to be high. On the other hand, when both proteins are detected, and particularly when only mutant Ang-1 is detected, it can be determined that the possibility of developing DR or the possibility of developing in the future is low.

  In addition, the preparation method of the said protein is not specifically limited, It can prepare by a conventionally well-known method from the test subject's all tissues, cells, etc. In particular, it is preferable to prepare from a tissue with a high expression level of Ang-1.

  In any of the methods (A) to (C), the subject is not particularly limited, but a person who has developed diabetes or a person who has developed DR is preferable.

  Furthermore, the sample to which the method for determining the onset or the possibility of the onset of DR according to the present invention is applied is only required to be a sample separated from a living body such as a human body, and is not limited to the examples exemplified above. Absent.

<3. Evaluation kit for the onset of DR or its possibility>
The determination kit of the onset of DR or the possibility of the onset according to the present invention (in other words, the DR diagnosis kit) only needs to contain at least a reagent capable of detecting the above mutation, and other configurations are particularly limited. It is not a thing. Examples of the reagent capable of detecting the mutation include a primer, a probe, and an antibody. These reagents may be contained alone or in a plurality of combinations. Furthermore, the determination kit can be obtained by combining other reagents in addition to the reagents exemplified above.

  Specifically, the kit for detecting the presence or absence of mutations on the genome and mRNA (cDNA) includes a primer designed to amplify the region containing the mutation position, and further detects the presence or absence of the mutation. Examples of the kit include a combination of one or more reagents necessary for detecting a mutation, such as a probe designed to be able to be used, a restriction enzyme, a reagent used for a base sequencing method such as the Maxam Gilbert method and the Sanger method. Such a reagent is appropriately selected and employed depending on the detection method employed, and examples thereof include dATP, dUTP, dTTP, dGTP, DNA synthase, and RNA synthase. Furthermore, an appropriate buffer solution and washing solution that do not hinder the detection of mutation may be included.

  In the case of a kit including a primer, the primer is not particularly limited as long as it can amplify the region including the mutation position. For example, the primer can be selected from the primers shown in SEQ ID NOs: 3 to 7 and used. .

  In the case of a kit containing a probe, the probe is not particularly limited as long as it can bind to the region containing the mutation position.

  Furthermore, examples of the kit for detecting the presence or absence of a mutation on a protein include a kit containing an antibody that recognizes only one of normal type Ang-1 and mutant type Ang-1.

By using these determination kits, the above <2. As described in the section of the method for determining the onset of DR or the possibility of its onset>, the diagnosis of DR (determination of the onset or the possibility of its onset) can be objectively and easily performed. The subject to which the determination kit for the onset of DR or the possibility of the onset is applied is not particularly limited, but a person who has developed diabetes or a person who has developed DR is preferable.

<4. DR onset prevention method and preventive drug>
As described above, normal Ang-1 is a protein that contributes to the onset of DR. On the other hand, normal Ang-1 has an insertion mutation of 1 amino acid, that is, mutant Ang-1 in which the above-mentioned glycine is inserted has an effect of suppressing the onset of DR. Therefore, supplementing the mutant Ang-1 having the glycine inserted therein with a normal Ang-1 is considered to be effective as a preventive method against the onset of DR. Therefore, the present invention may be used for (A) a method for preventing the onset of DR, or (B) a preventive agent for the onset of DR.

(A) Method for preventing the onset of DR The method for preventing the onset of DR according to the present invention is a method for treating a person having normal Ang-1 with a mutant Ang-1 or DNA encoding the mutant Ang-1, or It is preferable that the mutant Ang-1 is a method for preventing the onset of DR that complements a low molecular compound as an agonist of a receptor protein that serves as a ligand.

(B) Preventive drug against onset of DR A preventive drug against the onset of DR according to the present invention is a preventive drug used to prevent the onset of DR in a person having the above-mentioned normal type Ang-1, and is a mutant type It is a prophylactic agent against the onset of DR comprising, as a main component, Ang-1 or DNA encoding the mutant Ang-1 or a low molecular weight compound as an agonist of a receptor protein having the mutant Ang-1 as a ligand. Is preferred.

  In the preventive method and preventive agent for the onset of DR described above, the method for complementing mutant Ang-1 is not particularly limited. For example, a known protein expression system or gene transfer method can be used. Specifically, a method using an expression vector or a viral vector used for expressing a protein in mammalian cells can be mentioned.

  Examples of the receptor protein include a Tie-2 receptor. For example, Non-Patent Document 2 discloses that Ang-1 is a ligand for the Tie-2 receptor. Therefore, it is considered that administering a low molecular weight compound acting as an agonist of the Tie-2 receptor into the body by oral or intravenous injection is effective as a preventive method against the onset of DR. The low molecular compound referred to here includes proteins such as peptides.

  Further, the mutant Ang-1 or the DNA encoding the mutant Ang-1 and the low molecular compound as an agonist of the receptor protein are not only used alternatively as a preventive agent, but also used in combination. It is also possible to do.

  In addition, the subject to which the preventive method or preventive agent for the onset of DR according to the present invention is not particularly limited, but those who have developed diabetes are preferred.

  The present invention is not limited to the configurations exemplified above, and various modifications are possible within the scope shown in the claims, and the technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention.

  Although this invention is demonstrated more concretely based on an Example and FIGS. 1-5, this invention is not limited to this. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention. In the following examples, the presence or absence of mutations in DR-related genes was evaluated as follows.

[Evaluation method of presence or absence of mutation in Ang-1 gene]
(1) Sequence analysis EDTA blood was collected from the peripheral blood of the subject by a known method. Total RNA was isolated from the collected peripheral blood using an RNA isolation kit (GENTRA SYSTEMS, MN). A reverse transcription reaction with an Oligo dT primer was performed from the isolated total RNA by a known method to synthesize cDNA. And the amplification fragment | piece was obtained by RT-PCR method using the primer set from the exon 4 to 5 of Ang-1 gene. In addition, the primer used for said reaction is as showing below.
Sense primer F2-2: 5′-CCACCAACAACAGTGTCCTT-3 ′ (SEQ ID NO: 3)
Sense primer F2-4: 5′-CAACCTTGTCAATCTTTGC-3 ′ (SEQ ID NO: 4)
Antisense primer R1186: 5′-CAGCTTGATATACATCTGCACAG-3 ′ (SEQ ID NO: 7)
Moreover, the setting positions of the sense primers F2-2 and F2-4 and the antisense primer R1186 are as shown in FIG.

  Furthermore, the amplification conditions in the RT-PCR are as follows. First, fragment amplification by RT-PCR was performed using the synthetic cDNA as a template and the sense primer F2-2 and the antisense primer R1186 as primers.

The reaction solution in the RT-PCR is cDNA, 1 × PCR-Buffer II (Applied Biosystems), 1.5 mM MgCl ₂ , 0.2 mM dATP, dTTP, dGTP and dCTP, 200 nM each primer, 2.5 U Prepared to contain AmpliTaq Gold DNA-Polymerase (Applied Biosystems).

  Further, the RT-PCR reaction conditions were 95 ° C for 12 minutes for 1 cycle, 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1 minute for 30 cycles.

  After performing RT-PCR under the above conditions, 1 μl of the obtained PCR product was used as a template, and the above-mentioned reaction mixture composition and reaction conditions were used for PCR using the above-mentioned sense primer F2-4 and antisense primer R1186 as primers. The amplified fragment was obtained.

  As a result, the obtained amplified fragment was purified by ethanol precipitation. Next, a sample for sequence analysis was prepared from the purified amplified fragment by the dye terminator method using the sense primer F2-4 and BigDye Terminator (Applied Biosystems, CA). Thereafter, the samples were subjected to sequence analysis using a fluorescent DNA sequencer (ABI377, Applied Biosystems). By comparing the sequence of the amplified fragment determined by sequence analysis with the sequence of the mutant Ang-1 gene shown in SEQ ID NO: 1, the presence or absence of a mutation in the Ang-1 gene was evaluated (right side of FIG. 2). (See the upper and middle sections). When the sequence pherogram (waveform signal) is duplicated from the base number 805, it is determined that the mutant Ang-1 gene and the normal Ang-1 gene are present in heterogeneity (see the lower right side of FIG. 2). .

  In FIG. 2, “− / −”, “mt / mt”, and “mt / −” indicate that the normal Ang-1 gene is homozygous and the mutant Ang-1 gene is homozygous, respectively. And when the normal Ang-1 gene and the mutant Ang-1 gene are detected in a heterogeneous manner.

(2) Chain length analysis An amplified fragment was obtained in the same manner as in the case of the sequence analysis described above. However, in the chain length analysis, the sense primer F2-4 used was labeled with TET fluorescence. Chain length analysis was performed using a solution containing the amplified fragment obtained by diluting the final PCR reaction product 10-fold. For the chain length analysis, 1 μl of the above solution containing the amplified fragment is used as a sample, and 3 μl of a formamide solution containing 0.3 μl of 5 mg / ml Blue Dextran, 2.5 mM EDTA and molecular weight marker GS500 TAMRA (Applied Biosystems) is used as a sample. This was performed using a sequencer.

  In the chain length analysis, the presence or absence of a mutation in the Ang-1 gene was determined based on whether a 101-base or 98-base peak appeared (see the upper left and middle in FIG. 2). In addition, when peaks of both 101 bases and 98 bases appeared, it was determined to be heterogeneous (see the lower left part of FIG. 2).

[Example 1]
The above sequence analysis and chain length analysis were performed using peripheral blood collected from DR patients by the above method. FIG. 3 shows a sequence analysis result of a region including the mutation position. As shown in FIG. 3, it was found that there are two types of Ang-1 gene, a three-base insertion type and a three-base deletion type. The figure (left) shows the analysis result of the 3-base insertion type, and the figure (right) shows the analysis result of the 3-base deletion type. That is, it can be confirmed that the 3-base insertion type has a 3-base insertion of “GGT” (see FIG. 3 (left)), and the 3-base deletion type has a deletion of these 3 bases. Was confirmed (see FIG. 3 (right)).

  Of these, SEQ ID NO: 2 shows a 3-base insertion type gene sequence. In this three-base insertion type, as shown in SEQ ID NO: 2, three bases “GGT” are inserted as the 805th to 807th bases in the sequence.

  In addition, as shown in SEQ ID NO: 1, the protein that is the translation product of the three-base insertion gene has glycine inserted as the 269th amino acid in the amino acid sequence. On the other hand, the 269th glycine is missing in the protein that is the translation product of the three-base deletion gene.

[Example 2]
FIG. 4 shows the results of detecting the presence or absence of the 3-base insertion mutation in the Ang-1 gene by the above method using peripheral blood collected from RA patients, DR patients, and healthy individuals.

  In FIG. 4, n represents the total number of various subjects, a person who has a normal Ang-1 gene homozygously (in the figure and hereinafter referred to as “− / −”), a normal Ang-1 gene and a mutation Those who have heterozygous type-1 gene-1 (in the figure and hereinafter referred to as “mt / −”), those who have the mutant Ang-1 gene as homozygous (in the figure and hereinafter, indicated as “mt / mt”) ) Is shown in each column. Furthermore, in parentheses, the ratio of each of the above-mentioned subjects among various subjects is shown.

As shown in FIG. 4, in healthy individuals, “− / −” was 11.9%, “mt / −” was 78.0%, and “mt / mt” was 10.1%. In RA patients, “− / −” was 5.1%, “mt / −” was 79.0%, and “mt / mt” was 24.5%. In DR patients, “− / −” was 25.8%, “mt / −” was 36.5%, and “mt / mt” was 37.9%. Furthermore, about the person who has a variant Ang-1 gene by homology, the significant difference between a healthy subject group, RA patient group, and DR patient group was analyzed by the chi-square test. As a result, the P value between the healthy group and the RA patient group was 10 ⁻² , and the P value between the healthy group and the DR patient group was 10 ⁻⁴ . That is, in the DR patients, there were significantly more people who had the normal Ang-1 gene homozygously.

Example 3
DR patients were examined for age, hemoglobin A _1c (HbA1c), fasting blood glucose (FBS), total cholesterol (T-Cho) / HDL cholesterol (HDL-C), and years after onset of diabetes. In addition, HbA1c, FBS, T-Cho, and HDL-C were measured according to a conventional method using a fasting blood sample. As a result, for each measurement item, there was no difference due to the presence or absence of the Ang-1 gene mutation.

  In FIG. 5, a person who has a normal Ang-1 gene homozygously (in the figure and hereinafter referred to as “− / −”), and has a normal Ang-1 gene and a mutant Ang-1 gene in heterogeneity. Measurement of the above-mentioned various measurement items among those who have the mutant Ang-1 gene homozygously (in the figure and hereinafter referred to as “mt / mt”) The results are shown in the respective columns.

  Note that the present invention is not limited to the configurations described above, and various modifications are possible within the scope of the claims, and technical means disclosed in different embodiments and examples respectively. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention.

  As described above, in the present invention, the possibility of the onset of DR can be predicted by detecting a mutation in Ang-1 or the Ang-1 gene. Therefore, it can be widely used not only in the field of diagnostic medicine typified by a method for determining the onset of DR or its possibility of onset, but also in the field of health medicine. Furthermore, by using the mutant Ang-1 or the mutant Ang-1 gene, it can be used as a method for preventing the onset of DR or a preventive drug.

It is the schematic which showed typically a part of cDNA of Ang-1 gene concerning this invention. In a present Example, it is a figure which shows the result of having performed the sequence analysis and chain length analysis of the area | region containing the 3 base deletion mutation position of Ang-1 gene. In a present Example, it is a figure which shows the result of having performed the sequence analysis of the area | region containing the 3 base insertion mutation position of Ang-1 gene. In a present Example, it is a table | surface which shows the result of having detected the presence or absence of the 3 base deletion mutation in an Ang-1 gene in RA patient, DR patient, and a healthy subject. In a present Example, it is a table | surface which shows the result of having investigated age, HbA1c, FBS, T-Cho / HDL-C, and years after onset of diabetes about DR patient.

Claims (10)

A method for determining the onset or possibility of onset of diabetic retinopathy using a sample isolated from a living body,
Detecting the presence or absence of a mutation in a polynucleotide encoding angiopoietin-1 protein, and
The onset of diabetic retinopathy or the possibility of the onset thereof characterized in that the mutation is a 3-base deletion mutation encoding glycine, the 269th amino acid of mutant angiopoietin-1 protein Judgment method. A method for determining the onset or possibility of onset of diabetic retinopathy using a sample isolated from a living body,
Detecting the presence or absence of a mutation in the angiopoietin-1 gene, and
The above mutation is a 3-base deletion mutation of “GGT” (G and T represent guanine and thymine, respectively), which is the 805th to 807th base in the translation region of the mutant angiopoietin-1 gene. A method for determining the onset of diabetic retinopathy or the possibility of the onset thereof. A method for determining the onset or possibility of onset of diabetic retinopathy using a sample isolated from a living body,
Detecting the presence or absence of a mutation in the angiopoietin-1 protein, and
A method for determining the onset or the possibility of the onset of diabetic retinopathy, wherein the mutation is a deletion mutation of glycine, the 269th amino acid of mutant angiopoietin-1 protein.   The determination of the onset of diabetic retinopathy or the possibility of the onset thereof characterized by using the method for determining the onset of diabetic retinopathy or the possibility of the onset thereof according to any one of claims 1 to 3. kit.   5. The onset of diabetic retinopathy or the onset possibility thereof includes at least one of a primer, a probe, and an antibody. 5. Onset of diabetic retinopathy according to claim 4, Evaluation kit for the possibility of onset.   6. The diabetic retinopathy according to claim 5, wherein the primer is a primer for amplifying a region containing the 805th to 807th bases in the translation region of the mutant angiopoietin-1 gene. Kit for determining the onset of or the likelihood of onset.   6. The onset of diabetic retinopathy according to claim 5 or the onset thereof, wherein the probe binds to a region containing the 805th to 807th bases in the translation region of the mutant angiopoietin-1 gene. The possibility of judging kit.   The antibody is only one of the mutant angiopoietin-1 protein or the normal angiopoietin-1 protein lacking glycine which is the 269th amino acid of the mutant angiopoietin-1 protein. The kit for determining the onset or possibility of onset of diabetic retinopathy according to claim 5, wherein:   A person having a normal angiopoietin-1 protein lacking the 269th glycine of the mutant angiopoietin-1 protein, the mutant angiopoietin-1 protein having glycine as the 269th amino acid, or A DNA encoding a mutant angiopoietin-1 protein or a diabetic retinopathy characterized in that the mutant angiopoietin-1 protein complements a low molecular compound as an agonist of a receptor protein as a ligand. How to prevent onset. A prophylactic agent used to prevent the development of diabetic retinopathy,
A subject having a normal angiopoietin-1 protein lacking the 269th glycine of the mutant angiopoietin-1 protein;
A mutant angiopoietin-1 protein having glycine as the 269th amino acid, a DNA encoding the mutant angiopoietin-1 protein, or a receptor protein having the mutant angiopoietin-1 protein as a ligand; A preventive agent for the development of diabetic retinopathy, characterized by comprising a low-molecular compound as an agonist as a main component.

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