WO2013103226A1

WO2013103226A1 - Marker for early diagnosis of diabetic retinopathy and usage for same

Info

Publication number: WO2013103226A1
Application number: PCT/KR2012/011806
Authority: WO
Inventors: 김영수; 유형곤; 김경곤; 김상진; 유지영
Original assignee: 서울대학교 산학협력단
Priority date: 2012-01-03
Filing date: 2012-12-28
Publication date: 2013-07-11
Also published as: KR20130079953A; KR101343286B1

Abstract

The present invention relates to a composition for diagnosing diabetic retinopathy and a kit for diagnosing diabetic retinopathy. Also, the present invention relates to an analysis method for providing information required to diagnose diabetic retinopathy. As mentioned above, by providing a marker for early diagnosis of diabetic retinopathy, the present invention enables measuring and comparing genes with reduced expression or an expression level of a protein thereof in a diabetic retinopathy patient, thereby allowing early diagnosis of diabetic retinopathy and meaningful prediction or identification of the progression of disease. The marker according to the present invention enables noninvasive diagnosis and thus would be very useful in early detection from home and general clinics when a simple and valid diagnosis method using a biomarker of diabetic retinopathy by means of blood or urine testing is developed, and can be advantageous in reducing medical costs on a national level.

Description

Marker for early diagnosis of diabetic retinopathy and its use

The present invention relates to a composition for diagnosing diabetic retinopathy and a kit for diagnosing diabetic retinopathy. The present invention also relates to an analysis method for providing information necessary for diagnosing diabetic retinopathy.

Diabetic retinopathy (DR) is one of the three major ocular blindness disorders with macular degeneration and glaucoma. About 25% of the 5 million diabetic patients in Korea are known to have this disease, which occurs in about 40% of patients over 40 years of age with diabetes, and accounts for more than 60% of the blindness of elderly people over 65 years old. According to the National Health Insurance Corporation, the number of patients with diabetic retinopathy increased by 34% in four years from 153,000 in 2005 to 204,000 in 2009.

Diabetes increases blood sugar, causing blood circulation disorders and metabolic disorders, leading to eye disease. Blood vessels that nourish the retina in the second half of the eye become narrowed or blocked, resulting in accumulation of waste in blood vessels and bleeding. Diabetic retinopathy (DR) is caused by abnormal neovascularization in this area, not functioning properly as a blood vessel, and easily burst and cause bleeding.

Diabetic retinopathy is divided into non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). Non-proliferative diabetic retinopathy (NPDR), which accounts for about 90% of diabetic retinopathy, is characterized by abnormal retinal vascular abnormalities, bleeding, and macular edema.If left untreated, blindness or proliferative diabetic retinopathy (PDR) Deepening in the back causes huge adverse effects on vision, so timely treatment is necessary. When abnormal neovascularization grows and gradually develops proliferative diabetic retinopathy, intraocular bleeding may occur and tractional retinal detachment may occur. Severe intraocular hemorrhage can lead to blindness.

Diabetic retinopathy (DR) is rarely diagnosed early due to little early symptoms, poor vision, poor focus, blurring, or glare. In spite of the adventitious treatment, many patients are suffering from severe disease and leading to blindness. However, more than 85% of patients can maintain visual acuity if they receive proper treatment at the early stage and thoroughly manage their blood sugar. Therefore, there is an increasing need for early detection and suppression of diabetic retina and early treatment for high risk groups. However, the etiology of diabetic retinopathy has not yet been accurately identified, and biomarkers for determining the extent of diabetic retinopathy are very limited. Therefore, by developing novel early diagnostic markers and antibodies with high specificity and sensitivity, the present inventors intended to invent a biomarker for early diagnosis of diabetic retinopathy in blood.

Against this background, the present inventors attempted to identify biomarkers in the blood of non-proliferative diabetic retinopathy (NPDR) patients in the early stages of diabetic retinopathy. NPDR patients were classified into mild NPDR, moderate NPDR, and severe NPDR, and 15 blood samples from each group were taken, and 15 blood of patients with diabetes but not diabetic retinopathy (DR) were collected. As a result of verifying the results by using the Triple Quadrupole LC-MS / MS Multiple Reaction Monitoring (MRM) test method, the present invention was completed by discovering six biomarkers that specifically reduce expression in MO NPDR. .

One object of the present invention is alpha-1-acid glycoprotein 2 (alpha-1-acid glycoprotein 2), alpha- which is a new diagnostic marker for diabetic retinopathy, which can effectively diagnose the early diagnosis and deepening of diabetic retinopathy. Alpha-1-antitrypsin, Apolipoprotein A2, Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin Gelsolin) is to provide a marker for diagnosing diabetic retinopathy at least one selected from.

Still another object of the present invention is to provide a composition for diagnosing diabetic retinopathy comprising an agent for measuring the level of mRNA or protein thereof of at least one gene selected from the above markers.

Another object of the present invention to provide a kit for diagnosing diabetic retinopathy comprising the composition.

Still another object of the present invention is to provide a method for providing information necessary for diagnosing diabetic retinopathy by using the composition or kit for diagnosing diabetic retinopathy.

As one embodiment for achieving the above object, the present invention Alpha-1-acid glycoprotein 2 (Alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (Alpha-1-antitrypsin), apolipoprotein Providing one or more markers for diagnosing diabetic retinopathy selected from Apolipoprotein A2, Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin do.

As used herein, the term "diagnosis" refers to identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether diabetic retinopathy develops. Preferably it is to determine whether the development of non-proliferative diabetic retinopathy (NPDR) in the early stages of diabetic retinopathy.

As used herein, the term "diagnostic marker" refers to a significant increase in gene expression or protein expression levels in individuals with non-proliferative diabetic retinopathy compared to normal controls (non-diabetic retinopathy) or proliferative diabetic retinopathy. Or organic biomolecules such as polypeptides or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), which exhibit reduced behavior.

Markers for diagnosing diabetic retinopathy for the purposes of the present invention are alpha-1-acid glycoprotein 2 (alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (alpha-1-antitrypsin), apolipoprotein A2 (Apolipoprotein) A2), Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin.

Diabetic retinopathy is divided into non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). Non-proliferative diabetic retinopathy is characterized by the absence of blood vessel development, and proliferative diabetic retinopathy is different in its mechanism, such as the development of blood vessels, and non-proliferative diabetic retinopathy does not necessarily deepen into proliferative diabetic retinopathy. For example, even a marker known as a diagnostic marker for proliferative diabetic retinopathy may not necessarily be used as a diagnostic marker for non-proliferative diabetic retinopathy.

The present inventors through the following demonstration alpha-1-acid glycoprotein 2 (Alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (Alpha-1-antitrypsin), apolipoprotein A2 (Apolipoprotein A2) , Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin were identified as markers for diagnosing diabetic retinopathy.

In one embodiment of the present invention, biomarkers that overexpress or underexpress diabetic retinopathy by analyzing plasma samples from MH (macular hole), PDR (proliferative diabetic retinopathy), and NPDR (non-proliferative diabetic retinopathy) individuals Excavated. Specifically, samples of PDR and MH individuals were analyzed to identify PDR-specific candidate markers, and based on this, seven NPDR-specific markers were finally discovered.

In another embodiment of the present invention, plasma samples of normal controls (non-diabetic retinopathy) and NPDR individuals were analyzed to establish the effectiveness of the seven NPDR specific markers identified above.

In one embodiment, the present invention provides alpha-1-acid glycoprotein 2, alpha-1-antitrypsin, apolipoprotein A2, A formulation for measuring mRNA or protein levels of one or more genes selected from Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin Provided is a composition for diagnosing diabetic retinopathy.

Gene information of the six proteins of the present invention are alpha-1-acid glycoprotein 2, GeneBank Accession No. AAA51549, Uniprot ID: P19652, alpha-1-antitrypsin (Alpha- 1-antitrypsin, GeneBank Accession No. AAA51547, Uniprot ID: P01009), Apolipoprotein A2 (Apolipoprotein A2, GeneBank Accession No. AAA51701, Uniprot ID: P02652), Apolipoprotein A4 (Apolipoprotein A4, GeneBank Accession No. AAA96731, Uniprot ID: P06727), Beta-2-glycoprotein 1 (GeneBank Accession No. AAA51766, Uniprot ID: P02749), and Gelsolin (Gelsolin, GeneBank Accession No. AAH26033, Uniprot ID: P06396) to be.

However, Alpha-1-acid glycoprotein 2, Alpha-1-antitrypsin, Apolipoprotein A2, Apolipoprotein A4 A4), Beta-2-glycoprotein 1, and Gelsolin have no known association with diabetes retinopathy.

Alpha-1-acid-glycoprotein 2 (Alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (Alpha-1-antitrypsin), apolipoprotein A2 (Apolipoprotein A2), apolipoprotein A4 (Apolipoprotein A4) ), Beta-2-glycoprotein 1, and gelsolin are all specific gene expression levels in MO NPDR individuals compared to normal controls (nondiabetic retinopathy) or The expression level of the protein is reduced. The diabetic retinopathy subject is preferably a non-proliferative diabetic retinopathy subject.

The term "mRNA expression level measurement" used in the present invention is to measure the amount of mRNA in the process of confirming the presence and expression of mRNA of the genes for diagnosing diabetic retinopathy in a biological sample to diagnose diabetic retinopathy. Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real-time reverse transcriptase (Real-time RT-PCR), RNase protection assay (RPA). assays, Northern blotting, DNA chips, etc., but are not limited to these.

The agent for measuring mRNA expression level of a gene is preferably a primer pair or probe, and since the nucleic acid information of the genes is known in GeneBank et al., Those skilled in the art will recognize primers or probes that specifically amplify specific regions of these genes based on the sequence. You can design

The term "measurement of protein expression level" used in the present invention is a process of confirming the presence and degree of expression of a protein expressed from a gene for diagnosing diabetic retinopathy in a biological sample in order to diagnose diabetic retinopathy. Antibodies, interacting proteins, ligands, nanoparticles, or aptamers that specifically bind to protein or peptide fragments of the gene can be used to determine the amount of protein, but specific for the protein or peptide fragment. All detection means with an affinity can be included and preferably the protein expression level itself is measured without the use of antibodies, interacting proteins, ligands, nanoparticles or aptamers.

The protein expression level measurement or comparative analysis methods include protein chip analysis, immunoassay, ligand binding assay, Matrix Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF), and Surface Enhanced Laser Desorption / Ionization Time (SELDI-TOF). analysis of radiation mass spectrometry, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation, two-dimensional electrophoresis, liquid chromatography-liquid chromatography Mass Spectrometry (LC-MS), Liquid Chromatography-Mass Spectrometry / Mass Spectrometry (LC-MS / MS), Western Blot, and Enzyme linked immunosorbentassay (ELISA), but are not limited thereto.

As used herein, the term "diabetic retinopathy" refers to a complication in which peripheral circulatory disorder occurs due to diabetes and a decrease in vision occurs due to a disorder in the microcirculation of the retina, and preferably may be non-proliferative diabetic retinopathy. .

Preferably the agent for measuring the mRNA level is alpha-1-acid glycoprotein 2 (Alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (Alpha-1-antitrypsin), Apolipoprotein A2 (Apolipoprotein A2) Primer pairs, probes that specifically bind to one or more genes selected from Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin. Or antisense nucleotides.

As used herein, the term "primer pair" includes primer pairs of all combinations of forward and reverse primers that recognize a target gene sequence, but is preferably a primer pair that provides assay results with specificity and sensitivity. . When the nucleic acid sequence of a primer is a sequence that is inconsistent with a non-target sequence present in a sample, it can give high specificity when the primer amplifies only a target gene sequence containing complementary primer binding sites and does not cause nonspecific amplification. .

As used herein, the term “probe” refers to a substance that can specifically bind to a target substance to be detected in a sample, and means a substance that can specifically confirm the presence of the target substance in the sample through the binding. do. The type of probe molecule is a material commonly used in the art, but is not limited. Preferably, the probe molecule may be a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a peptide, a polypeptide, a protein, an RNA, or a DNA. It is PNA. More specifically, the probes include those derived from or similar to organisms or produced in vitro as biomaterials, for example enzymes, proteins, antibodies, microorganisms, flora and fauna, neurons, DNA, and RNA. DNA may include cDNA, genomic DNA, oligonucleotides, RNA includes genomic RNA, mRNA, oligonucleotides, and examples of proteins may include antibodies, antigens, enzymes, peptides, and the like.

As used herein, the term “antisense” refers to a nucleotide in which an antisense oligomer hybridizes with a target sequence in RNA by Watson-Crick base pairing, allowing formation of mRNA and RNA: oligomeric heterodimers, typically within the target sequence. It refers to oligomers having a sequence of bases and a backbone between subunits. The oligomer may have exact sequence complementarity or approximate complementarity to the target sequence.

Preferably the agent for measuring the protein level is alpha-1-acid glycoprotein 2 (Alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (Alpha-1-antitrypsin), Apolipoprotein A2 (Apolipoprotein A2) ), An antibody that specifically binds to one or more protein or peptide fragments selected from Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin. , Interaction proteins, ligands, nanoparticles, or aptamers.

As used herein, the term “antibody” refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, the antibody is the alpha-1-acid glycoprotein 2 (Alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (Alpha-1-antitrypsin), apolipoprotein A2 (Apolipoprotein A2) Means an antibody that specifically binds to at least one protein selected from apolipoprotein A4, beta-2-glycoprotein 1, and gelsolin, Polyclonal antibodies, monoclonal antibodies and recombinant antibodies. Generating antibodies can be readily prepared using techniques well known in the art.

The antibodies of the present invention also include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 and Fv.

As used herein, the term "aptamer" refers to a biopolymer material that inhibits protein interaction through three-dimensional binding with a specific target protein in the form of DNA, RNA of a single, double helix, and various target molecules. It has the characteristic of binding to. Typically the aptamers may be small nucleic acids of 15-50 bases in length that are folded into defined secondary and tertiary structures, such as stem-loop structures. The aptamers preferably bind to the target high or low expression protein with a kd of less than 10 ⁻⁶ , 10 ⁻⁸ , 10 ⁻¹⁰ , or 10 ⁻¹² . Aptamers can bind to high or low expression proteins with very high specificity, and aptamers can be composed of multiple ribonucleotide units, deoxyribonucleotide units, or a mixture of two types of nucleotide residues. In addition, the aptamers of the present invention may further comprise one or more modified base, sugar or phosphate backbone units.

In still another aspect, the present invention provides a kit for diagnosing diabetic retinopathy comprising the composition for diagnosing diabetic retinopathy. Preferably, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit.

In addition, preferably, the diabetic retinopathy diagnostic kit may further include one or more other component compositions, solutions, or devices suitable for analytical methods.

Preferably, the diagnostic kit may be a diagnostic kit comprising essential elements necessary to perform reverse transcriptase. The reverse transcription polymerase kit contains each primer pair specific for the marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each gene, and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. It may also include primers specific for the nucleic acid sequence of the control gene. Other reverse transcriptase kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC- DEPC-water, sterile water, and the like.

Preferably it may be a diagnostic kit characterized in that it contains the necessary elements necessary to perform the DNA chip. The DNA chip kit may include a substrate on which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and a reagent, a preparation, an enzyme, or the like for preparing a fluorescent probe. The substrate may also comprise cDNA or oligonucleotide corresponding to the control gene or fragment thereof.

Also preferably, it may be a diagnostic kit characterized by including the necessary elements necessary to perform the ELISA. ELISA kits include antibodies, interacting proteins, ligands, nanoparticles or aptamers that specifically bind to the protein or peptide fragment. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity to other proteins. They are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. The ELISA kit may also include antibodies specific for interacting proteins, ligands, nanoparticles, aptamers or control proteins that specifically bind to the protein or peptide fragment. Other ELISA kits can bind reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg conjugated with the antibody) and substrates or antibodies thereof. Other materials and the like.

As another aspect, the present invention provides a method for providing information for diagnosing diabetic retinopathy using the diabetic retinopathy diagnostic composition or the diabetic retinopathy diagnostic kit.

Preferably, the information providing method is alpha-1-acid glycoprotein 2, alpha-1-antitrypsin from a biological sample isolated from a suspected diabetic retinopathy. ), Expression of one or more genes selected from Apolipoprotein A2, Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin. Measuring the level or expression level of the protein; And it may be a method of providing information for diagnosing diabetic retinopathy comprising comparing the expression level of the gene or the expression level of the protein with a normal control sample.

As used herein, the term "biological sample" includes tissues, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid, or urine, which differ in gene expression levels or protein expression levels due to the development of diabetic retinopathy. This is not restrictive.

Alpha-1-acid-glycoprotein 2 (Alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (Alpha-1-antitrypsin), apolipoprotein A2 (Apolipoprotein A2), apolipoprotein A4 (Apolipoprotein A4) ), Beta-2-glycoprotein 1, and gelsolin are all specific gene expression levels in MO NPDR individuals compared to normal controls (nondiabetic retinopathy) or Since the expression level of the protein is reduced, it is possible to provide information by diagnosing diabetic retinopathy when the level is decreased. Preferably the diabetic retinopathy is non-proliferative diabetic retinopathy.

Alpha-1-acid-glycoprotein 2 of the present invention, alpha-1-antitrypsin, apolipoprotein A2, apolipoprotein A4 ( Apolipoprotein A4), Beta-2-glycoprotein 1, and Gelsolin are all diabetes mellitus because they specifically reduce the level of gene expression or the expression of the protein in MO NPDR individuals. When the expression level decreases as compared with the non-diabetic retinopathy (control), it can be judged as medium-term non-proliferative diabetic retinopathy (MO NPDR) during the advanced stage of diabetic retinopathy.

Preferably, the expression level of the gene of the present invention can measure or compare the mRNA expression level.

The mRNA expression level measurement or comparison may use reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip, etc., but the present invention is limited thereto. It is not. Through the above measurement methods, it is possible to confirm the mRNA expression level of the normal control group and the mRNA expression level of the diabetic retinopathy patient, and to compare or determine the level of these expressions to diagnose or predict the onset of diabetic retinopathy.

Preferably the protein expression level of the present invention can be measured and compared using antibodies, interacting proteins, ligands, nanoparticles or aptamers that specifically bind to the protein or peptide fragment. The antibody and the protein of interest in the biological sample form an antigen-antibody complex, and a method of detecting the antibody is used.

As used herein, the term “antigen-antibody complex” means a combination of a protein antigen and an antibody that recognizes it to identify the presence or absence of the gene of interest in a biological sample. The detection of the antigen-antibody complex can be detected using methods as known in the art, such as spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbing, chemical and other methods.

More preferably, the protein expression level measurement and comparison in the present invention is characterized by measuring and comparing the protein expression level itself without using an antibody.

For the purpose of the present invention, the protein expression level measurement or comparative analysis methods include protein chip analysis, immunoassay, ligand binding assay, Matrix Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, Surface Enhanced Laser (SELDITOF) Desorption / Ionization Time of Flight Mass Spectrometry analysis, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass Liquid chromatography-Mass Spectrometry (LCMS), liquid chromatography-Mass Spectrometry / Mass Spectrometry (LC-MS / MS), western blot, and enzyme linked immunosorbentassay (ELISA), but are not limited thereto.

In a specific embodiment of the present invention, alpha-1-acid glycoprotein 2, alpha-1-antitrypsin, apolipoprotein A2, Apolipoprotein A2, To measure and compare the protein expression levels of Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin itself, the LC-MRM method is used. .

Specifically, the protein in the biological sample is passed through the LC analysis column with a concentration gradient of 5% to 85% for 30 minutes with a solution of 5% distilled water, 95% acetonitrile and 0.1% formic acid based on volume%. The concentration gradient was carried out because the resolution of a specific material may vary depending on the solution mixing ratio, and thus the range was selected for the optimal range for simultaneously separating various proteins.

In mass spectrometry, quantitation is carried out using MS / MS mode, Multiple reaction monitoring (MRM) or SRM (selected reaction monitoring). Whereas Selected Ion Monitoring (SIM) is a method of using ions generated by collisions once in the source portion of the mass spectrometer, MRM selects a particular ion one more time from the broken ion to detect another continuously connected source of MS. It is a method of using the ions obtained from the collision after passing through it once more. More specifically, in SIM, there is a problem in that the selected ions may interfere with quantification when the selected ions are ions that are also detected in plasma. On the other hand, in case of using MRM, even if ions with the same mass are broken once more, the molecular structure is different and tends to be differentiated. Can be obtained. Thus, by using the MRM mode for mass spectrometry it is possible to simultaneously analyze the desired materials with better analytical sensitivity.

Through the above analysis methods, it is possible to compare the protein expression level in the normal control group and the protein expression level in the diabetic retinopathy individual, and to determine whether there is a significant increase or decrease in the expression level of the diabetic retinopathy marker gene to the protein. Diagnosis can be made.

As described above, the present invention provides a marker for early diagnosis of diabetic retinopathy, thereby measuring and comparing the expression level of the reduced expression gene or its protein in patients with non-proliferative diabetic retinopathy. Can significantly predict or identify early diagnosis and degree of disease

1 is a flow chart schematically showing the experimental process of the present invention.

2 to 4 are tables showing 142 transitions for 61 proteins presented as marker candidates in the Examples of the present invention.

FIG. 5 is a graph showing the duration of T2DM and the level of HbA1c for 60 plasma samples from ANOVA analysis results. Black represents the NoDR group, green represents the MI NPDR group, blue represents the MO NPDR group, and red represents the SV NPDR group.

6 shows four protein markers of the present invention, Alpha-1-acid glycoprotein 2, Alpha-1-antitrypsin, and apolipoprotein A2. A2), a box-plot showing the expression patterns of each of Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin will be.

7 is a graph showing an interactive plot and an ROC curve of alpha-1-acid glycoprotein 2 according to an embodiment of the present invention.

8 is a graph showing an interactive plot and ROC curve of alpha-1-antitrypsin according to an embodiment of the present invention.

9 is a graph showing an interactive plot and ROC curve of apolipoprotein A2 according to an embodiment of the present invention.

10 is a graph showing an interactive plot and ROC curve of apolipoprotein A4 according to an embodiment of the present invention.

11 is a graph showing an interactive plot and ROC curve of beta-2-glycoprotein 1 according to an embodiment of the present invention.

12 is a graph showing an interactive plot and ROC curve of gelsolin (Gelsolin) according to an embodiment of the present invention.

The present invention confirmed the efficacy of 61 protein marker candidates using MRM method in plasma samples of diabetic retinopathy patients. Specifically, the blood samples of non-proliferative diabetic retinopathy (NPDR) patients used in the present invention are 15 early non-proliferative diabetic retinopathy (mild NPDR, MI NPDR), 15 medium-term non-proliferative diabetic retinopathy (MO NPDR) In this study, 15 non-proliferative diabetic retinopathy (severe NPDR, SV NPDR) were used (total 45), and 15 blood samples of diabetic but not diabetic retinopathy (NoDR) patients were included. Six biomarkers that specifically reduce expression in NPDR was confirmed by MRM (Multiple Reaction Monitoring) experimental method using Triple Quadrupole LC-MS / MS to complete the present invention.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Samples and instruments used in the present invention are as follows.

Bicinchoninic acid (BCA, Cat. No: B9643) and copper (II) sulfate aqueous solution (Cat. No: C2284) were purchased from Sigma (St Louis, MO). Sequencing-grade modified trypsin (Cat.No: V5111, porcine) was purchased from Promega (Madison, WI), iodoacetamide (IAA, Cat.No: I1149), dithiothreitol for protein denaturation , DTT, Cat.No: 43815), Urea (urea, Cat.No: U6504), formic acid (FA, Cat.No: F0507), and trifluoroacetic acid (TFA, Cat.No: T62200) was purchased from Sigma. HPLC-grade methanol (HPLC-grade methanol, MeOH, Cat.No: UN1230), acetonitrile (acetonitrile, ACN, Cat.No: UN1648), and HPLC-grade water (Duksan, Seoul, Korea) Purchased). Sep-PAK Vac (1 cc, tC18 cartridges, Cat. No: WAT054960) used for peptide desalting was purchased from Waters (Milford, Mass.).

Peptide mixtures of beta-galactosidase (Cat. No: 4333606) from E. coli were purchased from Applied Biosystems (Foster City, CA). Pico Tip emitter (stock no: FS360-20-10-N-20-C12, ID in tip 10 μm, ID 20 μm, OD 360 μm, length 20 cm) and IntegraFrit capillary (stock no: IF360-75-50- N-5, ID 75 μm, OD 360 μm, length 50 cm) was purchased from New Objective (Woburn, MA). Sample vials (part no: LP1114-1265, 0.2-ml glass microinsert, silanized) were purchased from Interface Engineering (Seoul, Korea). In order to minimize uptake of protein or peptide, Protein LoBind tubes (Cat. No: 022431081, 1.5 ml) and low-retention tips (Rainin Instrument, Oakland, Calif.) Were used.

실시예 1: 당뇨망막병증 과발현 또는 저발현 단백질 발굴Example 1: Diabetic Retinopathy Overexpression or Low Expression Protein Discovery

A flow chart schematically illustrating the experimental process of the present invention is shown in FIG. 1.

In the present invention, PDR, which is a protein having a difference in expression through vitreous proteome analysis in patients with proliferative diabetic retinopathy (PDR) and macular hole (MH, non-diabetic control), in order to select a protein marker candidate group Specific candidate markers were identified. Next, the vitreous and corresponding plasma in patients with proliferative diabetic retinopathy (PDR), macular hole (MH), and non-proliferative diabetic retinopathy (NPDR), based on the degree of PDR protein expression obtained in the second study. Expression patterns of proteins in samples were analyzed by MRM.

As a result, we obtained a list of proteins that show differences in expression compared to macular hole (MH) and diabetic retinopathy patients (PDR), and additionally the lipoprotein family and complement element group, which are known to play an important role in diabetes. 142 transitions were determined for a total of 61 proteins including the complement component family and the SERPIN family (FIGS. 2-4).

In this study, mild NPDR, moderate NPDR, and severe NPDR were analyzed according to the control group (diabetic but not diabetic retinopathy) and non-proliferative diabetic retinopathy. Decrease Biomarkers Six biomarkers were identified (Table 1).

In the following Table 1, the AUC value is a calculated value derived from the ROC curve.

Table 1

No.	Uniprot ID	AUC	Protein name
One	P19652	0.796	Alpha-1-acid glycoprotein 2
2	P01009	0.778	Alpha-1-antitrypsin
3	P02652	0.778	Apolipoprotein A2
4	P06727	0.751	Apolipoprotein A4
5	P02749	0.747	Beta-2-glycoprotein 1
6	P06396	0.733	Gelsolin

실시예 2: 환자 선정 및 혈장 채취Example 2: Patient Selection and Plasma Collection

Plasma samples from 45 patients with non-proliferative diabetic retinopathy (NPDR) early in diabetic retinopathy (NPDR) and samples from control patients (diabetic but not diabetic retinopathy, NoDR) were obtained for LC MS / MS test samples. Clinical characteristics of the 45 patients with non-proliferative diabetic retinopathy (NPDR) and control patients are shown in Table 2 below. The patients were divided into three groups: mild, moderate, and severe.

(NoDR: group without diabetic retinopathy, MI NPDR: Mild NPDR, MO NPDR: Moderate NPDR, SV NPDR: Severe NPDR)

TABLE 2

group	Gender (female / male)	age	DM elapsed (years)	HbA1c (%)	Plasma concentration (µg / µl)	CV of plasma concentration (%)
NoDR	7/8	63.8 ± 9.5	7.9 ± 5.4	7.5 ± 1.6	63.8	9.1
MI NPDR	7/8	61.4 ± 6.7	12.3 ± 6.0	7.4 ± 1.3	61.4	9.4
MO NPDR	8/7	60.6 ± 9.9	15.9 ± 7.7	7.4 ± 1.1	60.6	7.9
SV NPDR	9/6	61.8 ± 9.1	11.8 ± 6.4	7.8 ± 0.8	61.8	9.7

In order to check the variance between the four groups (NoDR, MI NPDR, MO NPDR and SV NPDR), statistical analyzes of four classifications (gender, age, DM age, HbA1c) were performed. Pearson chi-square test was performed on gender and F-test was performed on age, DM progression, and HbA1c. Each test statistics, degree of freedom (df), p-value (p-value) and the resulting values are shown in Table 3 below.

As a result, HbA1c levels, representing the amount of sugar molecules reflecting the blood glucose mean over the last two to three months, increased by 7.4% in all groups, and there was no difference between groups in the ANOVA analysis.

TABLE 3

Classification	Test statistic	df	p -value	result	Analysis method
gender	1.6276	3	0.6531	No differences between groups	Pearson chi-square test
age	2.52	3	0.0675	No differences between groups	F-statistic
DM elapsed time	3.83	3	0.0148	MO vs. NoDR difference p -value = 0.0014	F-statistic
HbA1c	0.3	3	0.8232	No differences between groups	F-statistic

From the results of ANOVA analysis of Table 3, the duration of T2DM and the level of HbA1c of 60 plasma samples were graphically represented as shown in FIG. 5. According to the ANOVA analysis of Table 3, there was no specific variation between the four groups except for the type 2 diabetes course (A in FIG. 5) between NoDR and MO NPDR. In addition, there was no specific difference in variance between groups (B in FIG. 5) for HbA1c. In FIG. 5, black represents NoDR group, green represents MI NPDR group, blue represents MO NPDR group, and red represents SV NPDR group.

To identify the differences in diabetes treatment among the 44 groups (NoDR, MI NPDR, MO NPDR and SV NPDR), the statistical analysis of three categories: insulin, hyperglycemic drugs, statins for hyperlipidemia, and whether hypertension drugs were taken Was performed. A Fisher exact test was conducted for insulin and statins, and a Pearson chi-square test for hypertension therapy. The p-values for each analysis result are shown in Table 4 below.

Table 4

group	insulin		Statins		Hypertension
group	Taking	Unused	Taking	Unused	Taking	Unused
NoDR
	9	6	8	6	3	12
MI NPDR	10	5	7	3	One	10
MO NPDR	8	7	7	6	5	10
SV NPDR	7	8	5	10	4	11
Statistical analysis method	Pearson chi-square test		Fisher exact test		Fisher exact test
p -value	0.7155		0.3239		0.4966

As a result, differences among the four groups (NoDR, MI NPDR, MO NPDR, and SV NPDR) for the three items for the treatment of diabetes, hyperglycemia treatment (insulin), hyperlipidemia treatment (statin), and hypertension treatment, were significant for each item. No difference was found. Table 4

The data indicate that the plasma samples taken from the four groups have a relatively homogeneous diabetes background, and the present inventors believe that the difference in the expression level of the target protein is primarily due to the difference in stages of diabetic retinopathy. The hypothesis can be hypothesized.

Each plasma sample was analyzed using conventional methods (Park, J .; Kwon, H .; Kang, Y .; Kim, Y., Proteomic analysis of O-GlcNAc modifications derived from streptozotocin and glucosamine induced beta-cell apoptosis.J Biochem Mol Biol 2007 , 40, (6), 1058-68.). Specifically, blood was collected with a K2-EDTA-coated 10-ml tube (BD Sciences, NJ, P / N number: 367525) and centrifuged at 3000 g for 10 minutes at 4 ° C. Each plasma sample was divided into 50 μl samples and stored at -80 ° C.

실시예 3: 혈장 시료의 전처리Example 3: Pretreatment of Plasma Samples

Plasma samples were quantified using Bradford, of which 200 μg of plasma was taken and denatured into 100 μl 6 M Urea. It was then reduced to 1.7 μl 200 mM DTT and then alkylated with 6.7 μl 200 mM iodoacetic acid. 500 μl of distilled water was added thereto to dilute urea to 1 M or less, and then trypsin was treated at a ratio of 50: 1 (protein: trypsin, w / w) to make the denatured protein into a peptide. After overnight storage in a shaker at 37 ° C., 50 μl of 0.1% trifluoroacetic acid (TFA) was added to terminate the reaction. Denatured peptides were desalted using Sep-PAK tC18 cartridge (50 mg) and lyophilized with 2 mL 60% acetonitrile and 0.1% TFA solution. This was dissolved in 300 μl Solution A (95% distilled water, 5% acetonitrile, 0.1% formic acid), to which an internal standard beta-galactosidase peptide (residues 954-962, GDFQFNISR) was added. ) 50 fmol was spiked and analyzed by MRM.

실시예 4: Transition의 선정Example 4: Selection of Transition

In order to select the transitions of the proteins, MS / MS analysis was performed on the proteins selected in the excavation study. Based on this, a representative peptide for each protein was selected (Q1 transition), and the highest intensity ion (Q3) was selected among the fragmentation ions generated by electrically breaking the peptide. Two peptides per protein were selected and two fragmentation ions per peptide were selected to determine Q1 / Q3 as four transitions for one protein. Transitions were selected using the MRM Initiated Detection and Sequencing (MIDAS) workflow program (MRM Pliot, version 2.0, Applied biosystems, USA) for transitions that are difficult to select experimentally due to low peaks. Transitions that were not captured by the MIDAS workflow program were selected by selecting peptides with high observed numbers using the Peptide Atlas database.

실시예 5: LC 및 MRMExample 5: LC and MRM

LC used MDLC nanoflow Tempo LC of MDS. For peptide separation, C18 resin with a diameter of 3 μm and a pore size of 200 mm 3 was directly filled using a fused sillica capillary column having a length of 15 cm and an internal diameter of 100 μm. Peptide samples were injected by direct injection method, 1.0 μl was injected directly into the analitical column without passing through the trap column. The flow rate was 400 nl / min. Equilibrate the column with Solution A (95% distilled water, 5% acetonitrile, 0.1% formic acid) for 10 minutes and then 5% to 85% for 30 minutes with Solution B (5% distilled water, 95% acetonitrile, 0.1% formic acid). The peptide was eluted through a concentration gradient of 85% for 5 minutes.

Mass spectrometry was monitored in MRM mode for transitions to selected proteins using 4000 QTrap instrument, Applied Biosystems' hybrid triple quadrupole / linear ion trap. Ion voltage was used at 2000 Volt and the resolution at Quadruple 1 (Q1) and Quadruple 3 (Q3) was set in units. The dwell time for the transition was set to 20 milliseconds so that the total cycle time was 2.5 seconds. Neubulizing gas was used in 5 units, and heater temperature was set at 150 ℃ for analysis. To demonstrate batch-to-batch variation, 50 fmole beta-galactosidase peptide (beta-galactosidase peptide, Transition 542.3 / 636.3) spiked into each sample was also monitored at the same time. The MS run time was run for 60 minutes in time synchronization with the LC and the MS and LC were run using Analyst 2.1.2.

실시예 6 : 데이터 분석Example 6 Data Analysis

For relative quantification, beta-galatosidase peptide (Transition 542.3 / 636.3) was used to quantify MRM at 8 concentration points of blank, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, 100.0 fmol. A standard curve was determined. The individual MRM results were generated by extracting the ion extraction chromatography (XIC) of the MRM transitions using MultiQuant (AppliedBiosystems, ver1.0), calculating the peak area of each transition, and plotting it over time. It was. The area of each XIC peak is normalized to the XIC peak area of the beta-galatosidase peptide (Transition 542.3 / 636.3), an internal standard. Was performed. For statistical analysis, ROC (Receiver Operating Characteristic) Curve and Interactive plot were prepared using MedCalc (MedCalc Software, Belgium, vesion 11.3.3) and ANOVA (Analysis of variance) statistical analysis was performed. Sigma Plot (Systat Software Inc, USA, version 10.1) was used for some plotting and t-test analysis. Multivariable Analysis was performed to correlate many target proteins in classifying disease groups. The algorithm used was PLS-DA (Partial Least Square Discriminant Analysis) and SIMCA-P + (Umetrics, USA, demo version) software.

Clinically, SV NPDR causes bleeding on the surface of the retina, resulting in symptoms of decreased vision in the patient, resulting in diagnosis by ophthalmic examination. Therefore, early diagnosis of SV NPDR using markers is meaningless, and patients with SV NPDR were excluded from expression pattern analysis for diagnostic marker discovery.

Expression patterns of the six protein marker candidates in the present invention were observed in a box-plot form using NoDR, MI NPDR and MO NPDR groups, and are shown in FIG. 6. As a result of the analysis of the expression pattern, as shown in Figure 6, it can be seen that the six proteins of the present invention specifically decreases the expression in the MO NPDR group of the three groups, thus these proteins are used to diagnose the early stage It can be seen that it can be used as an indicator.

As a result of the analysis, six proteins having significant differences among the proteins having different expressions in NPDR patients compared to NoDR patients were identified, and ROC curves and interactive plots of the proteins are shown in FIGS. 7 to 12.

7 to 12 show alpha-1-acid glycoprotein 2, alpha-1-antitrypsin, apolipoprotein A2, and apolipo Interactive plots and ROC curves of protein A4, beta-2-glycoprotein 1, and gelsolin, respectively, with specific reductions in MO NPDR. By showing the results, it was confirmed that it can be used as a diagnostic marker specific for intermediate non-proliferative diabetic retinopathy (MO NPDR).

Sensitivity and specificity of each interactve plot of FIGS. 7 to 12 are expressed as "Sens" and "Spec", and are expressed as% values. The blue circle, on the other hand, represents the standardized concentration of each protein in NoDR against MO NPDR. In addition, the AUC value of each latent marker is shown in each ROC curve.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

The marker of the present invention enables a non-invasive diagnosis, if a diagnostic method using a simple and effective biomarker of diabetic retinopathy, such as blood, urine tests, etc. is developed, will be very useful for early detection in home and general clinic For example, medical costs can be reduced from a national perspective.

Claims

Alpha-1-acid glycoprotein 2, alpha-1-antitrypsin, apolipoprotein A2, apolipoprotein A4 , Beta-2-glycoprotein 1 (Beta-2-glycoprotein 1), and gelsolin (Gelsolin) composition for diagnosing diabetic retinopathy comprising an agent for measuring the mRNA or protein level of one or more genes selected from the group consisting of .
The method of claim 1,

The diabetic retinopathy is a non-proliferative diabetic retinopathy diabetic retinopathy diagnostic composition.
The method of claim 1,

The agent for measuring the mRNA level of the gene is a primer pair, a probe or an antisense nucleotide that specifically binds to the gene diabetic retinopathy diagnostic composition.
The method of claim 1,

The agent for measuring protein levels comprises diagnosing diabetic retinopathy comprising antibodies, interacting proteins, ligands, nanoparticles, or aptamers that specifically bind to the protein or peptide fragment.
Kit for diagnosing diabetic retinopathy comprising a composition according to any one of claims 1 to 4.
The method of claim 5,

The kit may be a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit. Kit for diagnosing diabetic retinopathy.
Alpha-1-acid glycoprotein 2, alpha-1-antitrypsin, apolipoprotein A2, apolipoprotein A4 (alpha-1-acid glycoprotein 2) from biological samples. Measuring the expression level of the at least one gene selected from the group consisting of Apolipoprotein A4), beta-2-glycoprotein 1, and gelsolin or the expression level of the protein; And

Method for providing information for diagnosing diabetic retinopathy comprising comparing the expression level of the gene or the expression level of the protein with a control sample.
The method of claim 7, wherein

Comparing the expression level of the gene or the expression level of the protein with the expression level of the gene of the control subject sample or the expression level of the protein, and determining that the expression is increased or decreased as non-proliferative diabetic retinopathy. How to provide information to diagnose the condition.
The method of claim 8,

The expression level of the gene is an mRNA expression level of the gene providing information for diagnosing diabetic retinopathy.
The method of claim 9,

Said mRNA level measurement is a reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting, or a DNA chip providing information for diagnosing diabetic retinopathy.
The method of claim 7, wherein

The method for measuring protein expression level provides information for diagnosing diabetic retinopathy by using antibodies, interacting proteins, ligands, nanoparticles, or aptamers that specifically bind to the protein or peptide fragment. .
The method of claim 7, wherein

The protein expression level is measured or compared by protein chip analysis, immunoassay, ligand binding assay, Matrix Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, and Surface Enhanced Laser Desorption / Ionization Time of Flight Mass (SELDI-TOF). Spectrometry analysis, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation, two-dimensional electrophoresis, liquid chromatography-Mass Spectrometry , LC-MS), LC-MS / MS (liquid chromatography-Mass Spectrometry / Mass Spectrometry), Western blot, and ELISA (enzyme linked immunosorbentassay) is carried out using a method selected from the group consisting of diabetic retinopathy How to provide information for diagnosis.