WO2020190081A1 - Blood marker for diagnosing major blindness-causing eye diseases, and diagnostic method using same - Google Patents
Blood marker for diagnosing major blindness-causing eye diseases, and diagnostic method using same Download PDFInfo
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- WO2020190081A1 WO2020190081A1 PCT/KR2020/003848 KR2020003848W WO2020190081A1 WO 2020190081 A1 WO2020190081 A1 WO 2020190081A1 KR 2020003848 W KR2020003848 W KR 2020003848W WO 2020190081 A1 WO2020190081 A1 WO 2020190081A1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/16—Ophthalmology
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/16—Ophthalmology
- G01N2800/164—Retinal disorders, e.g. retinopathy
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/16—Ophthalmology
- G01N2800/166—Cataract
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/16—Ophthalmology
- G01N2800/168—Glaucoma
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis
Definitions
- the present invention relates to a blood marker for diagnosing major eye diseases causing blindness and a diagnostic method using the same, and more particularly, to age related macular degeneration (AMD), diabetic retinopathy, cataract, and It relates to a marker for diagnosing one or more blindness-causing eye diseases selected from the group consisting of glaucoma, and a composition, kit, and a method for diagnosing blindness-causing eye diseases using the same.
- AMD age related macular degeneration
- cataract cataract
- ALD age related macular degeneration
- a marker for diagnosing one or more blindness-causing eye diseases selected from the group consisting of glaucoma, and a composition, kit, and a method for diagnosing blindness-causing eye diseases using the same.
- Vision is the most important sense in our body. As chronic diseases such as obesity, diabetes, and hypertension increase with the recent aging, the number of patients with eye diseases that can cause blindness, such as macular degeneration, diabetic retinopathy, glaucoma, and cataract, is increasing. In particular, the elderly with visual impairments are at increased risk of death due to traffic accidents or falls, and may experience emotional problems as it is difficult to live independently.
- age-related macular degeneration is a representative eye disease caused by various changes in the macular, the central part of the retina, which occurs mainly in adults over 50 years of age. It is the leading cause of blindness in adults and the incidence rate increases with age. Looking at the prevalence of age-related macular degeneration in Korea based on the National Health and Nutrition Survey, 11.7% of the population aged 60 to 69 and 18% of the population aged 70 or older in Korea are affected by age-related macular degeneration.
- the macula refers to the central part of the nervous tissue called the retina, and it is responsible for central vision because photoreceptor cells that respond to light stimuli are concentrated.
- Age-related macular degeneration is a disease that causes visual impairment due to loss of photoreceptor cells in the macular region with age.
- AMD is a degenerative disease affecting the retina, retinal pigment epithelium (RPE), Bruch's membrane, and choroid.
- RPE retinal pigment epithelium
- AMD can be divided into a dry or non-exudative (dry) form and a wet or exudative (exudative) form.
- the non-exudative form refers to a lesion in the retina, such as drusen or atrophy of the retinal pigment epithelium. It usually does not cause severe vision loss, but it can develop into a wet form.
- the exudative form is when choroidal neovascularization grows under the retina. These new blood vessels cause exudate and bleeding in the macula, damaging the photoreceptors, thereby lowering central vision, and most of them lead to legal blindness of 0.1 or less if not treated.
- the exudative form of macular degeneration is very rapid, and vision often deteriorates rapidly within a few weeks. Early detection of age-related macular degeneration is very important because, in general, once vision impairment begins, previous vision cannot be restored in many cases. Early detection is possible through regular ophthalmological examinations by an ophthalmologist.If age-related macular degeneration is suspected by ophthalmic examination including fundus examination, detailed ophthalmic examinations such as fluorescein angiography and optical coherence tomography should be performed to confirm the diagnosis. I can.
- Diabetic retinopathy is a typical complication of diabetes that occurs when microvessels in the retina are damaged.According to the Health Insurance Review and Assessment Service, the number of diabetic patients increased from about 2 million in 2012 to about 2.45 million in 2016. Compared to an increase of about 21%, the number of diabetic retinopathy patients increased from about 260,000 in 2012 to 336,000 in 2016, which increased to 38%, which is a larger increase than diabetes.
- Diabetic retinopathy occurs because abnormally new blood vessels are formed in this area, causing bleeding without normal blood vessel function.
- Diabetic retinopathy is classified into non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) according to its severity. In addition, NPDR is classified into mild, moderative and severe NPDR depending on the degree. Nonproliferative diabetic retinopathy shows retinal bleeding, microvascular perfusion, and cotton patches, but maintains good vision until late. In proliferative diabetic retinopathy, new blood vessels are formed in the retina, and if these blood vessels rupture, it causes severe bleeding in the vitreous cavity. It is absorbed over time, but becomes a fibrous tissue, which in turn causes tractional retinal detachment and rebleeding. Leads to blindness.
- NPDR non-proliferative diabetic retinopathy
- PDR proliferative diabetic retinopathy
- Diabetic retinopathy is difficult to diagnose early because there are few initial symptoms, and when symptoms (vision loss, loss of focus, glare) are in progress, the disease has already progressed, and despite treatment (laser, vitreous surgery), it leads to blindness. There are many patients. However, the need for early detection and suppression of diabetic retinopathy and early treatment for high-risk groups is emerging, since it is possible to maintain visual acuity if appropriate treatment is received at the beginning of the onset and blood sugar management is thorough.
- Cataract is a disease in which a structure called a transparent lens becomes cloudy in the eye, and the light cannot pass properly, and the overall vision becomes hazy as if it was fogged.
- the main cause is aging, and cataracts develop as the lens becomes less transparent with age. Patients with early or intermediate cataracts may take medication or use eye drops to slow the progression. If the cataract has progressed a lot, surgery is performed to remove the lens and insert an artificial lens.
- Glaucoma is a disease that occurs when the optic nerve is depressed due to increased pressure in the eye or the blood supply is impaired and damage to the optic nerve progresses over time. 5.1% of adults over the age of 40 in Korea suffer from glaucoma, and even if the intraocular pressure is slightly high, they do not feel any abnormalities, but the optic nerve continues to deteriorate and the field of view becomes narrow. Once damaged, the optic nerve is difficult to recover, so it is very important to detect and treat glaucoma early.
- biomarkers that determine the occurrence or progression of blindness-causing eye diseases such as age-related macular degeneration, diabetic retinopathy, cataracts or glaucoma are very limited, and continuous research is required.
- the inventors of the present invention discovered a blood protein marker with high sensitivity and specificity, and used it in combination to improve the early diagnosis of blindness-causing eye diseases such as age-related macular degeneration, diabetic retinopathy, cataract or glaucoma, etc.
- ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2), LGALS3BP (galectin-3- binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin-1) and ZG16B (Zymogen Granule protein 16 homolog B) at least one marker selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts or glaucoma, such as blindness-causing eye disease diagnosis efficiency was confirmed to be excellent, and completed the present invention.
- ADAMTS-like protein 2 ADAMTS-like protein
- Another object of the present invention is to provide a diagnostic kit for blindness-induced eye disease using the marker for diagnosing blindness-induced eye disease.
- Another object of the present invention is to provide a method of providing information necessary for diagnosing blindness-causing eye diseases using the marker for diagnosing blindness-induced eye disease.
- Another object of the present invention is to provide a method for screening a substance for treating blindness-causing eye diseases.
- the present invention is ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2), LGALS3BP ( galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin-1) And ZG16B (Zymogen granule protein 16 homolog B) provides a marker for diagnosis of at least one type of blindness-causing eye disease selected from the group consisting of.
- ADAMTSL2 ADAMTS-like protein 2
- Cp Ceruloplasmin
- CFH complement factor H
- DDI2 Protein DD
- the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
- the present invention is ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2), LGALS3BP (galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin- 1) and ZG16B (Zymogen granule protein 16 homolog B) provides a composition for diagnosing blindness-causing eye diseases, comprising a substance for measuring the protein level or mRNA level of at least one marker for diagnosis of blindness-causing eye disease.
- the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
- the agent for measuring the mRNA level of the complex marker may be a primer pair, a probe, or an antisense nucleotide that specifically binds to the gene of the marker.
- the agent for measuring the protein level of the complex marker is an antibody, interacting protein, ligand, nanoparticles or aptamer that specifically binds to the protein or peptide fragment. (aptamer) may be included.
- the present invention provides a kit for diagnosing blindness-causing eye diseases including the composition for diagnosing blindness-causing eye diseases.
- the kit is RT-PCR (Reverse transcription polymerase chain reaction) kit, DNA chip kit, ELISA (Enzyme linked immunosorbent assay) kit, protein chip kit, rapid kit, or MRM ( Multiple reaction monitoring) kit.
- RT-PCR Reverse transcription polymerase chain reaction
- DNA chip kit DNA chip kit
- ELISA Enzyme linked immunosorbent assay
- protein chip kit protein chip kit
- rapid kit or MRM ( Multiple reaction monitoring) kit.
- ADAMTSL2 ADAMTS-like protein 2
- Cp Ceruloplasmin
- CFH complement factor H
- DDI2 Protein DDI1 homolog2
- FCN2 Ficolin 2
- IGFBP2 insulin like growth factor binding protein 2
- LGALS3BP galectin-3-binding protein
- MBL2 mannose-binding protein C
- PNLIP pancreatic triacylglycerol lipase
- SELE E-selectin
- SIGLEC14 Sialic acid-binding Ig-like) lectin 14
- THBS1 Thrombospondin-1
- ZG16B Zymogen granule protein 16 homolog B
- the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
- the method of providing information for diagnosing blindness-causing eye diseases includes the patient's age, hypertension, hyperlipidemia, smoking, diabetes, BMI (body mass index), Hb1AC test results, and One or more clinical information selected from the group consisting of cardiovascular disease may be additionally included and compared with the control group.
- the mRNA expression level is measured using reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip. You can do it.
- the measurement of the protein expression level is performed by using an antibody, interacting protein, ligand, nanoparticles, or aptamer that specifically binds to a protein or peptide fragment. Can be done.
- the protein expression level measurement is protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDITOF (Sulface Enhanced).
- Laser Desorption/Ionization Time of Flight Mass Spectrometry analysis, radioimmunoassay, radioactive immunity diffusion method, octeroni immunity diffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis method, 2D electrophoresis analysis, liquid chromatography- Liquid chromatography-Mass Spectrometry (LC-MS), liquid chromatography-Mass Spectrometry/ Mass Spectrometry (LCMS/MS), Western blot, enzyme linked immunosorbent assay (ELISA), multiple reaction monitoring (MRM), Parallel reaction monitoring (PRM), sequential windowed data independent acquisition of the total high-resolution (SWATH), selected reaction monitoring (SRM), or immune multiple reaction monitoring (iMRM) It can be done using.
- LC-MS liquid chromatography- Liquid chromatography-Mass Spectrometry
- LCMS/MS liquid chromatography-Mass Spectrometry/ Mass Spectrometry
- Western blot enzyme linked immunosorbent assay
- the method of providing information for diagnosing blindness-causing eye disease further comprises determining that the gene expression level or protein expression level of the marker is increased compared to the control group as a blindness-causing eye disease.
- step (b) may be performed by a statistical analysis method.
- the statistical analysis method may include a linear or nonlinear regression analysis method, a linear or nonlinear classification analysis method, a logistic regression method, and an Analysis of Variance; ANOVA), neural network analysis method, genetic analysis method, support vector machine analysis method, hierarchical analysis or clustering analysis method, hierarchical algorithm using decision tree or kernel principal component analysis method, Markov Blanket analysis method , Recursive feature elimination or entropy-basic regression feature elimination analysis method, forward floating search or rear floating search analysis method, and a combination thereof.
- ANOVA Analysis of Variance
- the present invention is (a) ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein) 2), LGALS3BP (galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin-1) and ZG16B (Zymogen granule protein 16 homolog B); treating a candidate therapeutic agent in a cell sample in which the mRNA or protein expression level for one or more markers selected from the group consisting of increased compared to the control group; And
- the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
- the method for diagnosing blindness-causing eye disease of the present invention, a diagnostic composition, a diagnostic kit, or a method for providing information necessary for diagnosis is a new immunological diagnostic tool using plasma of a patient, and has excellent sensitivity and plasma without using a biopsy. In addition to being able to easily analyze the target, it was confirmed that high diagnostic ability was shown by combining the quantitative value of the protein and basic clinical information of the diagnostic marker, and thus it can be usefully used for diagnosis of blindness-causing eye diseases.
- ⁇ means a very low or high outlier as a result of box and whisker plot analysis.
- FIG. 3 is data analyzed by statistically processing results of quantification values of (A) ADAMTSL2 representative for macular degeneration disease (AMD) and (B) IGFBP2 representative marker for diabetic retinopathy (DMR) by AUC and T-test.
- ASD macular degeneration disease
- DMR diabetic retinopathy
- Figure 4 is a data obtained by combining the quantitative value of the protein of a single marker and basic clinical information of all macular degeneration patients and analyzed by statistical processing using a T-test (AMD: age-related macular degeneration, Non AMD: non-macular degeneration).
- DMR diabetic retinopathy
- Non DMR non-diabetic retinopathy
- the present invention is ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2).
- ADAMTSL2 ADAMTS-like protein 2
- Cp Ceruloplasmin
- CFH complement factor H
- DDI2 Protein DDI1 homolog2
- FCN2 Fecolin 2
- IGFBP2 insulin like growth factor binding protein 2
- the present invention relates to a marker for diagnosing one or more blindness-causing eye diseases selected from the group consisting of.
- diagnosis means identifying the presence or characteristics of a pathological condition.
- diagnosis is to determine whether or not blindness-causing eye disease has occurred.
- diagnostic marker used in the present invention refers to a polypeptide showing a significant increase or decrease in gene expression level or protein expression level in an individual with blindness-causing eye disease compared to a normal control group (individuals other than blindness-causing eye disease) or It includes organic biomolecules such as nucleic acids (eg, mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.).
- nucleic acids eg, mRNA
- lipids lipids
- glycolipids glycoproteins
- sugars monosaccharides, disaccharides, oligosaccharides, etc.
- the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
- macular degeneration is classified into early AMD and late AMD. Since early macular degeneration does not develop blood vessels, and late macular degeneration is different in its mechanisms, such as blood vessel development, even a marker known as a late macular degeneration diagnostic marker cannot necessarily be used as an early macular degeneration diagnostic marker.
- the markers of the present invention can specifically diagnose both early and late macular degeneration.
- Diabetic retinopathy is classified into early nonproliferative diabetic retinopathy (NPDR) and late proliferative diabetic retinopathy (PDR) according to the degree of progression.
- Nonproliferative diabetic retinopathy is characterized by no blood vessel development, and proliferative diabetic retinopathy differs in its mechanism, such as blood vessel development, and nonproliferative diabetic retinopathy does not necessarily progress to proliferative diabetic retinopathy. Then, even a marker known as a diagnostic marker for proliferative diabetic retinopathy cannot necessarily be used as a diagnostic marker for non-proliferative diabetic retinopathy.
- the markers of the present invention can specifically diagnose both non-proliferative diabetic retinopathy and proliferative diabetic retinopathy.
- ADAMTSL2 (ADAMTS-like protein 2) is a member of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-like protein subfamily, and is a glycoprotein that binds the cell surface and the extracellular matrix.
- ADAMTSL2 interacts with LTBP1 (latent transforming growth factor beta binding protein 1), and LTBP1 protein is involved in the storage of TGF- ⁇ 1, an important growth factor that regulates cell growth and division, so ADAMTSL2 has the possibility of using TGF- ⁇ 1. Adjust.
- the ADAMTSL2 may preferably include the amino acid sequence of SEQ ID NO: 1, but the amino acid sequence of SEQ ID NO: 1 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
- Cp is the main protein that carries copper in the blood and plays an important role in iron metabolism. More than 95% of copper is present in the plasma of healthy people in the form of ceruloplasmin.
- the Cp may preferably include the amino acid sequence of SEQ ID NO: 2, but the amino acid sequence of SEQ ID NO: 3 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
- CFH complement factor H
- complement factor H is a glycoprotein that plays an essential role in maintaining the immune response by regulating complement activation. It binds to the same sugar chain structure on the cell surface and acts as a complement inhibitor to prevent complement activation and amplification.
- the CFH may preferably include the amino acid sequence of SEQ ID NO: 3, but the amino acid sequence of SEQ ID NO: 3 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
- DDI2 Protein DDI1 homolog2 is an internal peptide cleavage enzyme that activates nuclear respiratory factor 1 (Nrf1), which is involved in regulating cell growth and DNA replication, and compensates for proteasome degradation.
- Nrf1 nuclear respiratory factor 1
- the DDI2 may preferably include the amino acid sequence of SEQ ID NO: 4, but the amino acid sequence of SEQ ID NO: 4 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
- FCN2 (Ficolin 2) is a type of oligolectin and is composed of a short N-terminal partial-collagen-like domain and a fibrinogen-like domain. It is mainly expressed in the liver and is known to play an important role in the lectin pathway of the complement system by binding to N-acetylglucosamin in the bacterial cell wall and acting as an opsonin like a mannose binding protein.
- the FCN2 may preferably include the amino acid sequence of SEQ ID NO: 5, but the amino acid sequence of SEQ ID NO: 5 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
- IGFBP2 insulin like growth factor binding protein 2
- IGFBP2 insulin like growth factor binding protein 2
- IGFBP2 insulin like growth factor
- the IGFBP2 may preferably include the amino acid sequence of SEQ ID NO: 6, but the amino acid sequence of SEQ ID NO: 6 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
- LGALS3BP (galectin-3-binding protein) is a protein encoded by the LGALS3BP gene, and specifically binds to Mac-2 (human macrophage-associated lectin) and galactin 1 (galectin 1). LGALS3BP is known to increase in serum of cancer patients and HIV-infected patients, and is involved in immune responses associated with natural killer (NK) cells and lymphokine-activated killer (LAK) cytotoxicity.
- NK natural killer
- LAK lymphokine-activated killer
- the LGALS3B may preferably include the amino acid sequence of SEQ ID NO: 7, but the amino acid sequence of SEQ ID NO: 7 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
- Mannose-binding protein C is also referred to as mannose-binding lectin (MBL) or mannan-binding protein (MBP).
- MBL2 has an oligomer structure (400-700 kDa) and is composed of subunits containing three identical peptide chains consisting of approximately 30 kDa. It is produced by the liver in response to infection and is part of a number of other factors called acute stage proteins.
- the MBL2 may preferably include the amino acid sequence of SEQ ID NO: 8, but the amino acid sequence of SEQ ID NO: 8 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
- PNLIP pancreatic triacylglycerol lipase
- PNLIP pancreatic triacylglycerol lipase
- PNLIP is known to have a low serum concentration because it is secreted into the duodenum through the pancreatic duct system.However, when pancreatic functions such as pancreatitis or pancreatic adenocarcinoma are extremely destroyed, pancreatic enzymes including PNLIP are secreted into the serum. It is known that acute pancreatitis can be diagnosed by measuring.
- the PNLIP may preferably include the amino acid sequence of SEQ ID NO: 9, but the amino acid sequence of SEQ ID NO: 9 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
- SELE E-selectin
- CD62E CD62 antigen-like family member E
- ELAM-1 endothelial-leukocyte adhesion molecule 1
- LECAM2 leukocyte-endothelial cell adhesion molecule 2
- interleukin 1 ⁇ tumor necrosis factor.
- SELE is a cell adhesion molecule that is transiently expressed and induced in vascular endothelial cells activated by lipopolysaccharides with a peak of 4 to 12 hours.
- SELE is strongly expressed in vascular endothelial cells in inflammatory tissues and promotes invasion of these cells into the inflammatory site by mediating the phenomenon that neutrophils and monocytes roll over vascular endothelial cells. It is known to be involved in adhesion of cancer cells to vascular endothelial cells.
- the SELE may preferably include the amino acid sequence of SEQ ID NO: 10, but the amino acid sequence of SEQ ID NO: 10 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
- SIGLEC14 (Sialic acid-binding Ig-like lectin 14) is one of the subfamily of SIGLEC (Sialic acid-binding immunoglobulin-type lectins), and SIGLEC is a cell surface protein that binds to sialic acid and is mainly expressed on the surface of immune cells.
- the protein interaction between SIGLEC and sialic acid acts as a switch to turn on and off the immune system, and cancer cells are also known to acquire resistance to the immune response by using the SIGLEC-sialic acid reaction.
- the SIGLEC14 may preferably include the amino acid sequence of SEQ ID NO: 11, but the amino acid sequence of SEQ ID NO: 11 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
- THBS1 Thrombospondin-1
- thrombospondin-1 is one of the thrombospondin family and is a glycoprotein that inhibits neovascularization and tumorigenesis. It is known that it binds to proteases related to angiogenesis, such as plasminogen, urokinase, MMP, thrombin, and cathepsin, and regulates adhesion, migration, and growth of endothelial cells.
- the THBS1 may preferably include the amino acid sequence of SEQ ID NO: 12, but the amino acid sequence of SEQ ID NO: 12 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
- ZG16B (Zymogen granule protein 16 homolog B) is a pancreatic adenocarcinoma upregulated factor (PAUF) that binds to carbohydrates and is known to activate internal epithelial cells, angiogenesis, and permeability.
- PAUF pancreatic adenocarcinoma upregulated factor
- the ZG16B may preferably include the amino acid sequence of SEQ ID NO: 13, but the amino acid sequence of SEQ ID NO: 13 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
- the markers can be used to diagnose blindness-causing eye diseases such as age-related macular degeneration, diabetic retinopathy, cataract or glaucoma.
- the present invention is ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2) ), LGALS3BP (galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 ( Thrombospondin-1) and ZG16B (Zymogen granule protein 16 homolog B), a composition for diagnosing blindness-causing eye diseases comprising a substance for measuring the protein expression level of at least one marker for diagnosis of blindness-causing eye disease or its mRNA expression level It is about.
- the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
- the agent for measuring the mRNA level of the complex marker is characterized in that it is a primer pair, probe or antisense nucleotide that specifically binds to the gene of the marker, and nucleic acid information of the genes is known in GeneBank, etc. Can design these primer pairs, probes or antisense nucleotides based on the above sequence.
- the term "measurement of mRNA expression level" used in the present invention is a process of confirming the presence and expression of mRNA of genes for diagnosis of blindness-causing eye disease in a biological sample isolated from a patient suspected of blindness-causing eye disease in order to diagnose blindness-causing eye disease Measure the amount of mRNA.
- primer as used in the present invention is a fragment that recognizes a target gene sequence, and includes forward and reverse primer pairs, preferably, a primer pair that provides an analysis result having specificity and sensitivity.
- a primer that amplifies only the target gene sequence containing the complementary primer binding site and does not induce non-specific amplification can give high specificity. .
- probe used in the present invention refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in a sample through the binding. do.
- the type of probe is a material commonly used in the art and is not limited, but preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferred Hagi is PNA.
- the probe is a biomaterial that includes an organism-derived or similar thing or a thing produced in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and It may be RNA, DNA includes cDNA, genomic DNA, oligonucleotide, RNA includes genomic RNA, mRNA, oligonucleotide, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.
- antisense refers to a nucleotide base in which an antisense oligomer is hybridized with a target sequence in RNA by Watson-Crick base pairing, typically allowing the formation of an mRNA and RNA: oligomeric heterodimer within the target sequence. It means an oligomer having a sequence of and a backbone between subunits. Oligomers may have exact sequence complementarity or approximate complementarity to the target sequence.
- the agent for measuring the protein level of the complex marker is an antibody, interacting protein, ligand, nanoparticles, or aptamer that specifically binds to the protein or peptide fragment. can do.
- protein expression level measurement used in the present invention is a process of determining the presence and expression of a protein expressed from a gene of a marker for diagnosis of blindness-causing eye disease in a biological sample in order to diagnose blindness-causing eye disease.
- an antibody refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction.
- an antibody refers to an antibody that specifically binds to the complex biomarker for diabetic retinopathy of the present invention.
- the antibodies of the present invention include all of polyclonal antibodies, monoclonal antibodies and recombinant antibodies.
- the antibody can be easily prepared using techniques well known in the art.
- the polyclonal antibody can be produced by a method well known in the art, including the process of injecting the diabetic retinopathy marker protein antigen into an animal and collecting blood from the animal to obtain serum containing the antibody.
- Such polyclonal antibodies can be prepared from any animal such as goat, rabbit, sheep, monkey, horse, pig, cow, dog.
- the monoclonal antibody is a hybridoma method well known in the art (see the hybridoma method; Kohler and Milstein, European Journal of Immunology 6:511-519, 1976), or phage antibody library technology (Clackson et al, Nature, 352 :624-628, 1991; Marks et al, J. Mol . Biol. , 222:58, 1-597, 1991).
- the antibody prepared by the above method may be separated and purified using a method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.
- the antibody of the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules.
- the functional fragment of an antibody molecule means a fragment that has at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2 and Fv.
- the antibody of the present invention may be obtained commercially.
- PNA Protein Nucleic Acid
- DNA has a phosphate-ribose sugar backbone
- PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by a peptide bond, which greatly increases the binding power and stability to DNA or RNA, resulting in molecular biology.
- Diagnostic analysis and antisense treatment PNA is described in detail in Nielsen PE et al, Science , 254(5037):1497-500, 1991.
- aptamer is an oligonucleotide or peptide molecule, and general information of the aptamer is described in Bock LC et al. , Nature , 355(6360):5646, 1992; Hoppe-Seyler F and Butz K, J Mol Med ., 78(8):42630, 2000; Cohen BA et al. , Proc Natl Acad Sci USA ., 95(24):142727, 1998).
- the present invention relates to a kit for diagnosing blindness-induced eye diseases, including the composition for diagnosing blindness-induced eye diseases.
- the kit can be prepared by a conventional manufacturing method known in the art.
- the kit may include, for example, an antibody in a freeze-dried form, a buffer, a stabilizer, an inactive protein, and the like.
- the kit may further include a detectable label.
- detectable label refers to an atom or molecule that specifically detects a molecule containing a label among molecules of the same type without a label.
- the detectable label may be attached to an antibody, interacting protein, ligand, nanoparticle, or aptamer that specifically binds to the protein or fragment thereof.
- the detectable label may include a radionuclide, a fluorophore, or an enzyme.
- the kit can be used according to various immunoassays or immunostaining methods known in the art.
- the immunoassay or immunostaining method may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA, capture-ELISA, inhibition or competition analysis, sandwich analysis, flow cytometry, immunofluorescence staining, and immunoaffinity purification.
- 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.
- RT-PCR reverse transcription polymerase chain reaction
- ELISA enzyme linked immunosorbent assay
- MRM multiple reaction monitoring
- the kit can be used for mass spectrometry.
- the specific amino acid residues of the protein are myristoylation, isoprenylation, prenylation, glypiation, lipoylation, acylation, alkylation, methylation, demethylation, amidation, It may have modifications such as ubiquitination, phosphorylation, deamidation, glycosylation, oxidation, or acetylation.
- ADAMTSL2 ADAMTS-like protein 2
- Cp Ceruloplasmin
- CFH complement factor H
- DDI2 Protein DDI1 homolog2
- FCN2 Ficolin 2
- IGFBP2 insulin like growth factor binding protein 2
- LGALS3BP glycosylcholine
- MBL2 mannose-binding protein C
- PNLIP pancreatic triacylglycerol lipase
- SELE E-selectin
- SIGLEC14 Sialic acid-binding protein Measuring the mRNA or protein level of at least one marker for diagnosis of blindness-causing eye diseases selected from the group consisting of Ig-like lectin 14), THBS1 (Thrombospondin-1) and ZG16B (Zymogen granule protein 16 homolog B);
- Ig-like lectin 14 Ig-like lectin 14
- THBS1 Thrombospondin-1
- ZG16B Zymogen granule protein 16 homolog B
- (b) It relates to a method for providing information for diagnosing blindness-causing eye diseases comprising the step of comparing the mRNA or protein expression level with a control sample.
- the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
- a "biological sample” refers to a tissue, cell, blood, serum, plasma, saliva, cerebrospinal fluid, or urine with a difference in protein expression level or gene expression level due to the onset of blindness-causing eye disease.
- the method of providing information for the diagnosis of blindness-causing eye disease includes at least one clinical information selected from the group consisting of age, hypertension, hyperlipidemia, smoking, diabetes, BMI (body mass index), Hb1AC test results, and cardiovascular disease. It may further include.
- the method of providing information for diagnosing blindness-causing eye disease may further include determining that it is a blindness-causing eye disease if the gene expression level or protein expression level of the marker increases/decreases compared to the control group.
- the mRNA expression level in step (a) can be measured and compared using a primer pair, a probe, or an antisense nucleotide specifically binding to the gene of the complex marker.
- mRNA expression level measurement or comparative analysis method reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip, etc. can be used, but the present invention It is not limited to these.
- the mRNA expression level in the normal control group and the mRNA expression level in patients with blindness-causing eye disease can be confirmed, and the onset of blindness-induced eye disease can be diagnosed or predicted by comparing the level of these expression levels.
- the measurement of the protein expression level in step (a) may be measured and compared using antibodies, interacting proteins, ligands, nanoparticles, or aptamers that specifically bind to proteins or peptide fragments.
- Protein expression level measurement or comparative analysis methods include protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDITOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight).
- MALDI-TOF Microx Desorption/Ionization Time of Flight Mass Spectrometry
- SELDITOF Surface Enhanced Laser Desorption/Ionization Time of Flight
- Mass Spectrometry Analysis Radiation Immunoassay, Radioimmune Diffusion Method, Okteroni Immunity Diffusion Method, Rocket Immunoelectrophoresis, Tissue Immunostaining, Complement Fixation Analysis, 2D Electrophoresis Analysis, Liquid Chromatography-Mass Spectrometry, LC-MS), LCMS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blot, and ELISA (enzyme linked immunosorbentassay), but are not limited thereto.
- MRM multiple reaction monitoring
- PRM parallel reaction monitoring
- SWATH sequential windowed data independent acquisition of the total high-resolution
- SRM selected reaction monitoring
- iMRM immune multiple reaction monitoring
- MRM is a method of determining an exact fragment of a substance, breaking it in a mass spectrometer, selecting a specific ion from the broken ions once more, and obtaining the number using a detector connected in series.
- the protein or fragment thereof can be quantified using a mass spectrometer in the blood samples of normal individuals and individuals suspected of causing blindness.
- the comparison of the mRNA or protein expression level in step (b) can be analyzed using a statistical method or an algorithm to improve the accuracy of diagnosis, and a linear or nonlinear regression analysis method, an advance or nonlinear classification analysis method, and logistic Logistic regression, Analysis of Variance (ANOVA), neural network analysis method, genetic analysis method, support vector machine analysis method, hierarchical analysis or clustering analysis method, hierarchical algorithm or kernel principal component using decision trees ( Kernel principal component analysis method, Markov Blanket analysis method, recursive feature elimination or entropy-basic regression feature elimination analysis method, forward floating search or rear floating search analysis An analysis method selected from the group consisting of a method, and a combination thereof may be used.
- the statistical method uses a logistic regression analysis method, but is not limited thereto.
- 184 plasma samples were analyzed for quantitative verification of biomarkers for diagnosis of age-related macular degeneration among plasma proteins (Table 1), and quantitative verification of biomarkers for diagnosis of diabetic retinopathy was performed. For this, 155 plasma samples were analyzed (Table 2).
- ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2), LGALS3BP (galectin-3) -binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin-1) and ZG16B ( 13 target markers composed of zymogen granule protein 16 homolog B) were analyzed using multiple reaction monitoring (MRM), and as shown in Table 3, peptides for each marker were selected for quantitative analysis and SIS MRM-MS analysis was performed using.
- MRM multiple reaction monitoring
- a logistic regression model is used to input conversion information of the expression level of a single marker to confirm the diagnostic ability according to the quantification of a single marker to estimate a probability value classified as age-related macular degeneration or diabetic retinopathy. I did.
- biomarker expression level conversion information and clinical information conversion value are input using a logistic regression model to confirm the effect of improving diagnostic ability.
- the probability of classification as age-related macular degeneration or diabetic retinopathy was estimated.
- the 13 types of markers of the present invention are suitable for diagnosis of blindness-causing eye diseases, and it was confirmed that the diagnostic ability of blindness-causing eye diseases is increased by combining the single marker with clinical information of the diagnosis subject.
- ADAMTSL2 ADAMTS-like protein 2
- Cp Ceruloplasmin
- CFH complement factor H
- DDI2 Protein DDI1 homolog2
- FCN2 Ficolin 2
- IGFBP2 insulin like growth factor binding protein 2
- LGALS3BP galectin-3-binding protein
- MBL2 mannose-binding protein C
- PNLIP pancreatic triacylglycerol lipase
- SELE E-selectin
- SIGLEC14 Sialic acid-binding Ig-like lectin 14
- THBS1 Thrombospondin-1
- ZG16B Zymogen granule protein 16 homolog B
- the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
- the control group may be cells derived from an individual who does not have blindness-causing eye disease.
- Example 1 Selection of patients with eye disease causing blindness and collection of plasma
- Plasma samples from patients with age-related macular degeneration were collected with the approval of the Institutional Review Board of Seoul National University Bundang Hospital. A total of 184 plasma samples were analyzed for quantitative detection of biomarkers using plasma proteins, and the clinical characteristics of the analyzed target normal group (Non AMD) and age-related macular degeneration (AMD) disease group are shown in Table 1 below.
- Plasma samples from diabetic retinopathy patients were collected with the approval of the Institutional Review Board of Seoul National University Bundang Hospital. A total of 155 plasma samples were analyzed for quantitative detection of biomarkers using plasma proteins, and the clinical characteristics of the analyzed normal group (Non DMR) and diabetic retinopathy (DMR) disease group are shown in Table 2 below.
- ADAMTSL2 ADAMTS-like protein 2
- Cp Ceruloplasmin
- CFH complement factor H
- DDI2 Protein DDI1 homolog2
- FCN2 Ficolin 2
- IGFBP2 insulin like growth factor binding protein 2
- LGALS3BP galectin-3-binding protein
- MBL2 mannose-binding protein C
- PNLIP pancreatic triacylglycerol lipase
- SELE E-selectin
- SIGLEC14 Sialic acid-binding Ig-
- THBS1 Thrombospondin-1
- ZG16B Zymogen granule protein 16 homolog B
- a representative peptide having a specific charge-to-mass ratio (m/z) for 13 biomarker proteins is selected (Q1), and the peptide is broken by electric shock.
- the ion (Q3) having the highest strength was selected.
- At least one peptide with high sensitivity per protein was measured and injected into a mass spectrometer based on this to obtain an optimal value of fragmentation energy per transition, and three or more upper fragmented ions were selected based on the intensity (Table 3).
- SIS Stable-isotope labeled standard
- SIS peptide is a peptide obtained by substituting 13C and 15N for 12C and 14N in the amino acids of lysine (Lys, K) or arginine (Arg, R) at the C-terminus of the peptide. This has a difference in mass value from the endogenous peptide present in blood, but since it has the same sequence, the peptide hydrophobicity is the same, so it is eluted at the same retention time (RT) on the chromatogram.
- RT retention time
- Example 1 Each plasma obtained in Example 1 was used as it is, or 14 kinds of proteins (Albumin, IgG, Antitrypsin, IgA, Transferrin, Haptoglobin, Fibrinogen, Alpha2-Macroglobulin, etc.) exist in high amounts for more accurate protein quantification.
- Alpha1-Acid glycoprotein, IgM, Apolipoprotein AI, Apolipoprotein AII, Complement C3, Transthyretin) were removed.
- 14 kinds of proteins were removed using a MARS (Multiple affinity removal system, Agilent, USA) column according to the manufacturer's method, and the remaining proteins were eluted and used for analysis.
- MARS Multiple affinity removal system
- 2-carboxylethyltrisphosphine Tris(2-carboxyethyl)-phosphine, TECP
- 2-chloro 2-chloroacetamide
- Acetamide (2-chloroacetamide) was added and reacted at 25° C. for 1 hour to reduce and alkylate disulfide bonds.
- Rice seed enzyme was added so that the mass ratio of plasma protein to rice seed (wako) enzyme was 100:1, and reacted at room temperature for 4 hours.
- Example 2-1 a heavy-labeled peptide was added (spiking) as an internal standard material (SIS peptide) determined in Example 2-1 to perform MRM analysis.
- SIS peptide an internal standard material
- Nano ultra 2D plus (Eksigent), a triple quadrupole mass spectrometer, QTarp 5500 (SCIEX) was used to monitor the transition of each selected protein in a scheduled MRM mode.
- the peak area of the transition was calculated by processing raw data using Skyline (Mccoss lab, University of Washington, USA). Relative concentrations were compared for the endogenous/heavy labeled peptide using the peak area. Using the measured results, T-test and Area under the receiver operating characteristic (AUROC) values were generated to measure the predictive ability of each protein peptide. In order to confirm the predictive power combined with clinical information, the expression level conversion information and the degree of conversion of the clinical information in Table 1 or 2 are input using a logistic regression model to determine the probability value classified as a blindness-causing eye disease. Estimated. All statistical analyzes were performed using MedCal ver 17.1 (MedCalc).
- the body mass index (BMI), smoking, hyperlipidemia, hypertension, cardiovascular disease, and glycated hemoglobin (Hb1Ac) values were reflected in the clinical information.
- BMI body mass index
- Hb1Ac glycated hemoglobin
- the biomarker expression level conversion information measured in Example 2 was input to measure a probability value classified as age-related macular degeneration.
- Diagnosis of age-related macular degeneration according to a single marker type One 2 3 4 5 6 7 8 9 10 11 12 13 AUC 0.71 0.65 0.61 0.61 0.60 0.68 0.66 0.61 0.61 0.63 0.60 0.60 0.60
- the biomarker expression level conversion information measured in Example 2 was input to measure a probability value classified as diabetic retinopathy.
- Diabetic Retinopathy Diagnosis Ability by Single Marker Type One 2 3 4 5 6 7 8 9 10 11 12 13 AUC 0.60 0.67 0.58 0.57 0.63 0.63 0.67 0.67 0.61 0.58 0.64 0.58 0.58
- Example 4 For diagnosis of blindness-causing eye diseases Marker Confirmation of diagnostic ability according to the combination of analysis results and clinical information
- the conversion information of the amount of biomarker expression and the degree of conversion of the clinical information are input using a logistic regression model, The probability value to be classified was measured.
- Diagnosis of age-related macular degeneration by combining single marker and clinical information One 2 3 4 5 6 7 8 9 10 11 12 13 AUC 0.72 0.62 0.62 0.62 0.61 0.69 0.66 0.62 0.62 0.62 0.61 0.61
- the biomarker expression level conversion information and the clinical information conversion level are input using a logistic regression model and classified as diabetic retinopathy. The probability value to be obtained was measured.
- Diabetic retinopathy diagnosis ability by combining single marker and clinical information One 2 3 4 5 6 7 8 9 10 11 12 13 AUC 0.71 0.76 0.71 0.71 0.72 0.75 0.71 0.74 0.73 0.71 0.73 0.71 0.71 0.71 0.71
- the method for diagnosing blindness-causing eye diseases provided by the present invention, a diagnostic composition, a diagnostic kit, or a method for providing information necessary for diagnosis is a new immunological diagnostic tool using plasma of a patient, and has excellent sensitivity and does not use a biopsy.
- high diagnostic ability is shown by combining the quantitative value of the protein and basic clinical information of the diagnostic marker, and thus it can be usefully used for diagnosis of blindness-causing eye diseases.
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Abstract
The present invention relates to a blood marker for diagnosing major blindness-causing eye diseases, and a diagnostic method using same, and, more specifically, to: a marker for diagnosing at least one blindness-causing eye disease selected from a group consisting of age-related macular degeneration (AMD), diabetic retinopathy, cataracts, and glaucoma; a composition and a kit for diagnosing blindness-causing eye diseases by using same; and a method for diagnosing blindness-causing eye diseases. A marker for diagnosing blindness-causing eye diseases, a diagnostic composition, a diagnostic kit, or a method for providing information required for diagnosis, of the present invention, are novel immunological diagnostic tools using the plasma of patients, have excellent sensitivity and enable convenient analysis of plasma without using a biopsy, and have been confirmed to show a high diagnostic ability when protein quantitative values of the diagnostic marker and basic clinical information are combined and analyzed, thereby being effectively usable for the diagnosis of blindness-causing eye diseases.
Description
본 발명은 실명 유발 주요 안질환 진단용 혈액 마커 및 이를 이용한 진단 방법에 관한 것으로, 보다 상세하게는 나이관련황반변성(Age related macular degeneration, AMD), 당뇨망막병증(Diabetic retinopathy), 백내장(cataract) 및 녹내장(glaucoma)으로 구성된 군에서 선택된 하나 이상의 실명 유발 안질환을 진단하기 위한 마커 및 이를 이용한 실명 유발 안질환 진단용 조성물, 키트 및 실명 유발 안질환을 진단하는 방법에 관한 것이다.The present invention relates to a blood marker for diagnosing major eye diseases causing blindness and a diagnostic method using the same, and more particularly, to age related macular degeneration (AMD), diabetic retinopathy, cataract, and It relates to a marker for diagnosing one or more blindness-causing eye diseases selected from the group consisting of glaucoma, and a composition, kit, and a method for diagnosing blindness-causing eye diseases using the same.
시각은 우리 몸에서 가장 중요한 감각이다. 최근 고령화와 함께 비만과 당뇨, 고혈압 등 만성질환이 증가하면서 황반변성이나 당뇨망막병증, 녹내장, 백내장 등 실명을 유발할 수 있는 안질환 환자가 증가하는 추세이다. 특히 시각장애가 있는 노인은 교통사고나 낙상으로 인한 사망 위험이 높아지고 독립적인 생활이 어려워 정서적인 문제까지 겪을 수 있다.Vision is the most important sense in our body. As chronic diseases such as obesity, diabetes, and hypertension increase with the recent aging, the number of patients with eye diseases that can cause blindness, such as macular degeneration, diabetic retinopathy, glaucoma, and cataract, is increasing. In particular, the elderly with visual impairments are at increased risk of death due to traffic accidents or falls, and may experience emotional problems as it is difficult to live independently.
실명 유발 안질환 중에서 나이관련황반변성(age-related macular degeneration: AMD)은 50세 이상의 성인에서 주로 발생하는 망막의 중심부인 황반(macular)에 여러 가지 변화가 동반되어 생기는 대표적 안질환으로써, 선진국에서는 성인 실명의 가장 주된 원인이며 나이가 증가함에 따라 발생율이 증가한다. 국민건강영양조사를 근거로 국내 나이관련 황반변성의 유병률을 살펴보면 한국인에서 60 ~ 69세 인구의 11.7%, 70세 이상 인구의 18%가 나이관련 황반변성에 이환되어 있다. 황반은 망막이라고 하는 신경 조직의 중심 부위를 말하는데, 빛 자극에 반응하는 광수용체 세포가 밀집되어 있어서 중심 시력을 담당한다. 나이가 들어감에 따라 황반부 광수용체 세포가 소실되어 시력 장애를 일으키는 질환을 나이관련황반변성이라 한다. AMD는 망막, 망막색소상피층(RPE), 부르크막(Bruch's membrane), 맥락막에 영향을 미치는 퇴행성 질병이다.Among the eye diseases that cause blindness, age-related macular degeneration (AMD) is a representative eye disease caused by various changes in the macular, the central part of the retina, which occurs mainly in adults over 50 years of age. It is the leading cause of blindness in adults and the incidence rate increases with age. Looking at the prevalence of age-related macular degeneration in Korea based on the National Health and Nutrition Survey, 11.7% of the population aged 60 to 69 and 18% of the population aged 70 or older in Korea are affected by age-related macular degeneration. The macula refers to the central part of the nervous tissue called the retina, and it is responsible for central vision because photoreceptor cells that respond to light stimuli are concentrated. Age-related macular degeneration is a disease that causes visual impairment due to loss of photoreceptor cells in the macular region with age. AMD is a degenerative disease affecting the retina, retinal pigment epithelium (RPE), Bruch's membrane, and choroid.
AMD는 건성 또는 비삼출성(dry) 형태와 습성 또는 삼출성(exudative) 형태로 나눌 수 있다. 비삼출성 형태는 망막에 드루젠(drugen)이나 망막색소 상피의 위축과 같은 병변이 생긴 경우를 말한다. 이는 보통 심한 시력상실을 유발하지는 않지만 습성 형태로 발전할 수 있다. 삼출성 형태는 망막 밑에 맥락막 신생혈관이 자라는 경우이다. 이러한 신생혈관은 황반부에 삼출물, 출혈 등을 일으켜서 광수용체를 손상시키고 그에 따라 중심시력을 저하시켜, 치료를 하지 않을 경우 대부분 0.1 이하의 법적 실명을 초래한다. 삼출성 형태의 황반변성은 진행속도가 매우 빨라서 수 주 안에 시력이 급속히 나빠지는 경우가 많다. 나이관련 황반변성은 일반적으로 일단 시력장애가 시작되면 이전의 시력을 회복할 수 없는 경우가 많으므로 조기 발견이 매우 중요하다. 조기 발견은 안과의사에 의한 정기적인 안과검진을 통해서 가능하며 안저검사를 포함한 안과적 검진으로 나이관련 황반변성이 의심되면, 형광안저혈관조영술, 빛간섭단층촬영 등의 안과 정밀검사들을 시행하여 확진할 수 있다. AMD can be divided into a dry or non-exudative (dry) form and a wet or exudative (exudative) form. The non-exudative form refers to a lesion in the retina, such as drusen or atrophy of the retinal pigment epithelium. It usually does not cause severe vision loss, but it can develop into a wet form. The exudative form is when choroidal neovascularization grows under the retina. These new blood vessels cause exudate and bleeding in the macula, damaging the photoreceptors, thereby lowering central vision, and most of them lead to legal blindness of 0.1 or less if not treated. The exudative form of macular degeneration is very rapid, and vision often deteriorates rapidly within a few weeks. Early detection of age-related macular degeneration is very important because, in general, once vision impairment begins, previous vision cannot be restored in many cases. Early detection is possible through regular ophthalmological examinations by an ophthalmologist.If age-related macular degeneration is suspected by ophthalmic examination including fundus examination, detailed ophthalmic examinations such as fluorescein angiography and optical coherence tomography should be performed to confirm the diagnosis. I can.
당뇨망막병증(diabetic retinopathy, DR 또는 DMR)은 망막의 미세혈관이 손상되었을 때에 나타나는, 당뇨병의 대표적인 합병증으로, 건강 보험심사평가원에 따르면 당뇨병 환자는 2012년 약 200만 명에서 2016년 약 245만명으로 21% 정도 증가한데 비해 당뇨망막병증 환자수는 2012년 약 26만 명이었는데 2016년 33만 6000명으로 38%로 증가해, 당뇨병보다 증가폭이 컸다.Diabetic retinopathy (DR or DMR) is a typical complication of diabetes that occurs when microvessels in the retina are damaged.According to the Health Insurance Review and Assessment Service, the number of diabetic patients increased from about 2 million in 2012 to about 2.45 million in 2016. Compared to an increase of about 21%, the number of diabetic retinopathy patients increased from about 260,000 in 2012 to 336,000 in 2016, which increased to 38%, which is a larger increase than diabetes.
당뇨로 인해 혈당이 높아지면서, 지속적인 고혈당으로 인해 망막혈관의 미세순환에 장애가 일어나 안구의 후반부 망막에 영양을 공급하는 혈관이 좁아지거나 막혀서 노폐물이 축적됨 되어 출혈이 발생한다. 이 부위에 비정상적으로 신생 혈관이 생기면서 정상적인 혈관기능을 못하고 출혈을 일으킴으로써 당뇨망막병증이 발생하게 된다.As blood sugar rises due to diabetes, the microcirculation of retinal blood vessels is disturbed due to continuous high blood sugar, and the blood vessels supplying nutrients to the retina in the second half of the eye are narrowed or blocked, causing waste products to accumulate and bleeding. Diabetic retinopathy occurs because abnormally new blood vessels are formed in this area, causing bleeding without normal blood vessel function.
당뇨망막병증은 심화정도에 따라 비증식석 당뇨망막병증(non-proliferative diabetic retinopathy, NPDR)과 증식성 당뇨망막병증(proliferative diabetic retinopathy, PDR)로 구분한다. 또한 NPDR을 경우는 정도에 따라 mild, moderative 및 severe NPDR로 분류된다. 비증식성 당뇨망막병증은 망막의 출혈, 미세혈관류, 면화반등의 소견을 보이지만 늦게까지 좋은 시력을 유지한다. 증식성 당뇨망막병증은 망막에 신생혈관이 생성되고, 이 혈관이 파열하면 유리체강 내 심각한 출혈을 야기, 시간이 지나면 흡수되지만 섬유성 조직으로 변해 나중에는 견인성 망막박리 및 재출혈이 발생해 영구적은 실명을 일으키게 된다.Diabetic retinopathy is classified into non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) according to its severity. In addition, NPDR is classified into mild, moderative and severe NPDR depending on the degree. Nonproliferative diabetic retinopathy shows retinal bleeding, microvascular perfusion, and cotton patches, but maintains good vision until late. In proliferative diabetic retinopathy, new blood vessels are formed in the retina, and if these blood vessels rupture, it causes severe bleeding in the vitreous cavity. It is absorbed over time, but becomes a fibrous tissue, which in turn causes tractional retinal detachment and rebleeding. Leads to blindness.
당뇨망막병증은 초기증상이 거의 없어 조기 진단이 어렵고, 증상(시력저하, 초점상실, 눈부심)을 보일 땐, 이미 병이 진행된 상태이며, 치료(레이저, 유리체 수술)에도 불구하고 심화되어 실명에 이르는 환자가 많다. 다만 발병 초기에 적절한 치료를 받고, 혈당관리를 철저히 하면 시력 유지가 가능하기에, 당뇨망막병증의 조기발견과 억제 및 고위험군에 대한 조기 치료에 대한 필요성이 대두되고 있다. Diabetic retinopathy is difficult to diagnose early because there are few initial symptoms, and when symptoms (vision loss, loss of focus, glare) are in progress, the disease has already progressed, and despite treatment (laser, vitreous surgery), it leads to blindness. There are many patients. However, the need for early detection and suppression of diabetic retinopathy and early treatment for high-risk groups is emerging, since it is possible to maintain visual acuity if appropriate treatment is received at the beginning of the onset and blood sugar management is thorough.
백내장은 눈 속에 투명한 수정체라는 구조물이 혼탁해져 빛을 제대로 통과하지 못해 안개가 낀 것처럼 시야가 전체적으로 뿌옇게 되는 질환이다. 주된 원인은 노화이며, 나이가 들면 수정체의 투명성이 떨어지면서 백내장이 생긴다. 초기 또는 중기 백내장 환자는 약물을 복용하거나 안약을 활용해 진행을 늦출 수 있다. 백내장이 많이 진행된 경우에는 수정체를 제거하고 인공수정체를 삽입하는 수술을 한다.Cataract is a disease in which a structure called a transparent lens becomes cloudy in the eye, and the light cannot pass properly, and the overall vision becomes hazy as if it was fogged. The main cause is aging, and cataracts develop as the lens becomes less transparent with age. Patients with early or intermediate cataracts may take medication or use eye drops to slow the progression. If the cataract has progressed a lot, surgery is performed to remove the lens and insert an artificial lens.
녹내장은 눈의 압력이 높아져 시신경이 눌리거나 혈액 공급에 장애가 생겨 시간이 지남에 따라 시신경 손상이 진행하여 발생하는 질환이다. 우리나라 40세 이상 성인의 5.1%가 녹내장에 시달리며, 안압이 다소 높더라도 별다른 이상을 느끼지 못하지만 시신경은 계속 나빠지고 시야가 좁아지게 된다. 한번 손상된 시신경은 회복이 어렵기 때문에, 녹내장을 조기에 발견하여 치료하는 것이 매우 중요하다.Glaucoma is a disease that occurs when the optic nerve is depressed due to increased pressure in the eye or the blood supply is impaired and damage to the optic nerve progresses over time. 5.1% of adults over the age of 40 in Korea suffer from glaucoma, and even if the intraocular pressure is slightly high, they do not feel any abnormalities, but the optic nerve continues to deteriorate and the field of view becomes narrow. Once damaged, the optic nerve is difficult to recover, so it is very important to detect and treat glaucoma early.
하지만, 나이관련황반변성, 당뇨망막변증, 백내장 또는 녹내장 등과 같은 실명 유발 안질환의 발생 여부 또는 진행정도를 판단하는 바이오마커는 매우 한정적으로 이에 대한 지속적인 연구가 필요한 실정이다.However, biomarkers that determine the occurrence or progression of blindness-causing eye diseases such as age-related macular degeneration, diabetic retinopathy, cataracts or glaucoma are very limited, and continuous research is required.
이에, 본 발명자들은 민감도 및 특이성이 높은 혈액 단백질 마커를 발굴하고, 이를 복합적으로 사용하여 나이관련황반변성, 당뇨망막병증, 백내장 또는 녹내장 등과 같은 실명 유발 안질환의 조기 진단능을 높이고자 노력한 결과 ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B) 으로 구성된 군에서 선택된 1종 이상의 마커가 나이관련황반변성, 당뇨망막병증, 백내장 또는 녹내장 등과 같은 실명 유발 안질환 진단 효율이 우수한 것을 확인하고, 본 발명을 완성하였다.Accordingly, the inventors of the present invention discovered a blood protein marker with high sensitivity and specificity, and used it in combination to improve the early diagnosis of blindness-causing eye diseases such as age-related macular degeneration, diabetic retinopathy, cataract or glaucoma, etc. ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2), LGALS3BP (galectin-3- binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin-1) and ZG16B (Zymogen Granule protein 16 homolog B) at least one marker selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts or glaucoma, such as blindness-causing eye disease diagnosis efficiency was confirmed to be excellent, and completed the present invention.
본 발명의 목적은 실명 유발 안질환 진단용 조성물을 제공한다.It is an object of the present invention to provide a composition for diagnosing blindness-causing eye diseases.
본 발명의 다른 목적은 상기 실명 유발 안질환 진단용 마커를 이용한 실명 유발 안질환진단 키트를 제공하는 데 있다.Another object of the present invention is to provide a diagnostic kit for blindness-induced eye disease using the marker for diagnosing blindness-induced eye disease.
본 발명의 또 다른 목적은 상기 실명 유발 안질환 진단용 마커를 이용한 실명 유발 안질환 진단에 필요한 정보를 제공하는 방법을 제공하는 데 있다.Another object of the present invention is to provide a method of providing information necessary for diagnosing blindness-causing eye diseases using the marker for diagnosing blindness-induced eye disease.
본 발명의 또 다른 목적은 실명 유발 안질환 치료용 물질의 스크리닝 방법을 제공한다.Another object of the present invention is to provide a method for screening a substance for treating blindness-causing eye diseases.
상기 목적을 달성하기 위해, To achieve the above object,
본 발명은 ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)으로 구성된 군에서 선택된 1종 이상의 실명 유발 안질환 진단용 마커를 제공한다.The present invention is ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2), LGALS3BP ( galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin-1) And ZG16B (Zymogen granule protein 16 homolog B) provides a marker for diagnosis of at least one type of blindness-causing eye disease selected from the group consisting of.
본 발명의 바람직한 일실시예에 있어서, 상기 실명 유발 안질환은 나이관련황반변성, 당뇨망막병증, 백내장 및 녹내장으로 구성된 군에서 선택된 하나 이상일 수 있다.In a preferred embodiment of the present invention, the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
또한, 본 발명은 ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)으로 구성된 군에서 선택된 1종 이상의 실명 유발 안질환 진단용 마커의 단백질 수준 또는 그의 mRNA 수준을 측정하는 물질을 포함하는 실명 유발 안질환 진단용 조성물을 제공한다In addition, the present invention is ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2), LGALS3BP (galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin- 1) and ZG16B (Zymogen granule protein 16 homolog B) provides a composition for diagnosing blindness-causing eye diseases, comprising a substance for measuring the protein level or mRNA level of at least one marker for diagnosis of blindness-causing eye disease.
본 발명의 바람직한 일실시예에 있어서, 상기 실명 유발 안질환은 나이관련황반변성, 당뇨망막병증, 백내장 및 녹내장으로 구성된 군에서 선택된 하나 이상일 수 있다.In a preferred embodiment of the present invention, the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
본 발명의 바람직한 다른 일실시예에 있어서, 상기 복합 마커의 mRNA 수준을 측정하는 제제는 상기 마커의 유전자에 특이적으로 결합하는 프라이머쌍, 프로브 또는 안티센스 뉴클레오타이드일 수 있다.In another preferred embodiment of the present invention, the agent for measuring the mRNA level of the complex marker may be a primer pair, a probe, or an antisense nucleotide that specifically binds to the gene of the marker.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 복합 마커의 단백질 수준을 측정하는 제제는 상기 단백질 또는 펩타이드 단편에 특이적으로 결합하는 항체, 상호작용 단백질, 리간드, 나노입자(nanoparticles) 또는 압타머(aptamer)를 포함할 수 있다. In another preferred embodiment of the present invention, the agent for measuring the protein level of the complex marker is an antibody, interacting protein, ligand, nanoparticles or aptamer that specifically binds to the protein or peptide fragment. (aptamer) may be included.
또한, 본 발명은 상기 실명 유발 안질환 진단용 조성물을 포함하는 실명 유발 안질환 진단용 키트를 제공한다. In addition, the present invention provides a kit for diagnosing blindness-causing eye diseases including the composition for diagnosing blindness-causing eye diseases.
본 발명의 바람직한 일실시예에 있어서, 상기 키트는 RT-PCR(Reverse transcription polymerase chain reaction) 키트, DNA 칩 키트, ELISA (Enzyme linked immunosorbent assay) 키트, 단백질 칩 키트, 래피드(rapid) 키트 또는 MRM(Multiple reaction monitoring) 키트일 수 있다.In a preferred embodiment of the present invention, the kit is RT-PCR (Reverse transcription polymerase chain reaction) kit, DNA chip kit, ELISA (Enzyme linked immunosorbent assay) kit, protein chip kit, rapid kit, or MRM ( Multiple reaction monitoring) kit.
또한, 본 발명은 (a) 환자의 생물학적 시료로부터 ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)으로 구성된 군에서 선택된 1종 이상의 실명 유발 안질환 진단용 마커의 mRNA 또는 단백질 수준을 측정하는 단계; 및 In addition, the present invention (a) ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2), LGALS3BP (galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like) lectin 14), THBS1 (Thrombospondin-1) and ZG16B (Zymogen granule protein 16 homolog B) measuring the mRNA or protein level of at least one marker for diagnosis of blindness-causing eye disease; And
(b) 상기 mRNA 또는 단백질 발현 수준을 대조군 시료와 비교하는 단계를 포함하는 실명 유발 안질환 진단을 위한 정보제공 방법을 제공한다. (b) It provides a method of providing information for diagnosing blindness-causing eye diseases comprising the step of comparing the mRNA or protein expression level with a control sample.
본 발명의 바람직한 일실시예에 있어서, 상기 실명 유발 안질환은 나이관련황반변성, 당뇨망막병증, 백내장 및 녹내장으로 구성된 군에서 선택된 하나 이상일 수 있다.In a preferred embodiment of the present invention, the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
본 발명의 바람직한 다른 일실시예에 있어서, 상기 실명 유발 안질환 진단을 위한 정보제공 방법은 환자의 나이, 고혈압 여부, 고지혈증 여부, 흡연 여부, 당뇨 여부, BMI (body mass index), Hb1AC 검사결과 및 심혈관 질환 여부로 구성된 군에서 선택된 1종 이상의 임상정보를 추가로 포함하여 대조군과 비교할 수 있다. In another preferred embodiment of the present invention, the method of providing information for diagnosing blindness-causing eye diseases includes the patient's age, hypertension, hyperlipidemia, smoking, diabetes, BMI (body mass index), Hb1AC test results, and One or more clinical information selected from the group consisting of cardiovascular disease may be additionally included and compared with the control group.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 mRNA 발현 수준 측정은 역전사효소 중합효소반응, 경쟁적 역전사효소 중합효소반응, 실시간 역전사효소 중합효소반응, RNase 보호 분석법, 노던 블랏팅 또는 DNA 칩을 이용하여 수행할 수 있다. In another preferred embodiment of the present invention, the mRNA expression level is measured using reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip. You can do it.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 단백질 발현 수준 측정은 단백질 또는 펩타이드 단편에 특이적으로 결합하는 항체, 상호작용 단백질, 리간드, 나노입자(nanoparticles) 또는 압타머(aptamer)를 이용하여 수행할 수 있다.In another preferred embodiment of the present invention, the measurement of the protein expression level is performed by using an antibody, interacting protein, ligand, nanoparticles, or aptamer that specifically binds to a protein or peptide fragment. Can be done.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 단백질 발현 수준 측정은 단백질 칩 분석, 면역측정법, 리간드 바인딩 어세이, MALDI-TOF(Matrix Desorption/Ionization Time of Flight Mass Spectrometry)분석, SELDITOF(Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry)분석, 방사선 면역분석, 방사 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 조직면역 염색, 보체 고정 분석법, 2차원 전기영동 분석, 액상 크로마토그래피-질량분석(liquid chromatography-Mass Spectrometry, LC-MS), LCMS/MS(liquid chromatography-Mass Spectrometry/ Mass Spectrometry), 웨스턴 블랏, ELISA(enzyme linked immunosorbent assay), 다중 반응 모니터링(multiple reaction monitoring: MRM), 병행 반응 모니터링(parallel reaction monitoring: PRM), sequential windowed data independent acquisition of the total high-resolution(SWATH), 선택 반응 모니터링(selected reaction monitoring: SRM) 또는 면역 다중 반응 모니터링(immuno multiple reaction monitoring: iMRM)을 이용하여 수행할 수 있다. In another preferred embodiment of the present invention, the protein expression level measurement is protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDITOF (Sulface Enhanced). Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioactive immunity diffusion method, octeroni immunity diffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis method, 2D electrophoresis analysis, liquid chromatography- Liquid chromatography-Mass Spectrometry (LC-MS), liquid chromatography-Mass Spectrometry/ Mass Spectrometry (LCMS/MS), Western blot, enzyme linked immunosorbent assay (ELISA), multiple reaction monitoring (MRM), Parallel reaction monitoring (PRM), sequential windowed data independent acquisition of the total high-resolution (SWATH), selected reaction monitoring (SRM), or immune multiple reaction monitoring (iMRM) It can be done using.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 실명 유발 안질환 진단을 위한 정보제공 방법은 마커의 유전자 발현 수준 또는 단백질 발현 수준이 대조군에 비해 증가하면 실명 유발 안질환이라고 판정하는 단계를 추가로 포함할 수 있다. In another preferred embodiment of the present invention, the method of providing information for diagnosing blindness-causing eye disease further comprises determining that the gene expression level or protein expression level of the marker is increased compared to the control group as a blindness-causing eye disease. Can include.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 (b) 단계는 통계적 분석 방법에 의해 수행될 수 있다.In another preferred embodiment of the present invention, step (b) may be performed by a statistical analysis method.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 통계적 분석 방법은 선형 또는 비선형 회귀 분석방법, 선형 또는 비선형 분류(classification) 분석방법, 로지스틱 회귀 분석방법(logistic regression), 분산분석(Analysis of Variance; ANOVA), 신경망 분석방법, 유전적 분석방법, 서포트 벡터 머신 분석방법, 계층 분석 또는 클러스터링 분석방법, 결정 트리를 이용한 계층 알고리즘 또는 커널 주성분(Kernel principal component) 분석방법, 마르코프 블랭킷(Markov Blanket) 분석방법, 회귀 특성 소거(recursive feature elimination) 또는 엔트로피-기본 회귀 특성 소거 분석방법, 전방 플로팅 서치(floating search) 또는 후방 플로팅 서치(floating search) 분석방법, 및 이들의 조합으로 구성되는 군으로부터 선택될 수 있다.In another preferred embodiment of the present invention, the statistical analysis method may include a linear or nonlinear regression analysis method, a linear or nonlinear classification analysis method, a logistic regression method, and an Analysis of Variance; ANOVA), neural network analysis method, genetic analysis method, support vector machine analysis method, hierarchical analysis or clustering analysis method, hierarchical algorithm using decision tree or kernel principal component analysis method, Markov Blanket analysis method , Recursive feature elimination or entropy-basic regression feature elimination analysis method, forward floating search or rear floating search analysis method, and a combination thereof. .
나아가, 본 발명은 (a) ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)으로 구성된 군에서 선택된 1종 이상의 마커에 대한 mRNA 또는 단백질 발현 수준이 대조군에 비해 증가된 세포 시료에 치료제 후보물질을 처리하는 단계; 및Further, the present invention is (a) ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein) 2), LGALS3BP (galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin-1) and ZG16B (Zymogen granule protein 16 homolog B); treating a candidate therapeutic agent in a cell sample in which the mRNA or protein expression level for one or more markers selected from the group consisting of increased compared to the control group; And
(b) 상기 후보 물질 처리후, 상기 마커의 mRNA 또는 단백질 발현 수준이 감소되었는지를 확인하는 단계를 포함하는 실명 유발 안질환 치료용 물질의 스크리닝 방법을 제공한다. (b) After the treatment of the candidate substance, it provides a method for screening a substance for treating blindness-causing eye diseases, comprising the step of determining whether the mRNA or protein expression level of the marker is decreased.
본 발명의 바람직한 일실시예에 있어서, 상기 실명 유발 안질환은 나이관련황반변성, 당뇨망막병증, 백내장 및 녹내장으로 구성된 군에서 선택된 하나 이상일 수 있다.In a preferred embodiment of the present invention, the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
본 발명의 실명 유발 안질환 진단용 마커, 진단용 조성물, 진단용 키트 또는 진단에 필요한 정보를 제공하는 방법은 환자의 혈장을 이용하는 새로운 면역학적 진단 도구로서, 민감도가 우수할 뿐만 아니라 생검을 이용하지 않고 혈장을 대상으로 간편하게 분석할 수 있을 뿐만 아니라, 진단용 마커의 단백질 정량값과 기본적인 임상정보를 결합하여 분석하면 높은 진단능을 보이는 것을 확인하였으므로, 실명 유발 안질환의 진단에 유용하게 사용될 수 있다.The method for diagnosing blindness-causing eye disease of the present invention, a diagnostic composition, a diagnostic kit, or a method for providing information necessary for diagnosis is a new immunological diagnostic tool using plasma of a patient, and has excellent sensitivity and plasma without using a biopsy. In addition to being able to easily analyze the target, it was confirmed that high diagnostic ability was shown by combining the quantitative value of the protein and basic clinical information of the diagnostic marker, and thus it can be usefully used for diagnosis of blindness-causing eye diseases.
도 1은 정상인의 혈장과 황반변성 환자의 혈장에서 LC-MRM 방법을 이용하여 측정된 13개의 단백질 정량값을 T-검정으로 통계처리 하여 분석한 데이터이다 (Non AMD: 비황반변성 환자, AMD: 황반변성). 도면에서 ○는 상자수염도(box and whisker plot) 분석 결과 매우 낮거나 높은 이상치(outlier)를 의미한다.1 is data obtained by statistically processing 13 protein quantification values measured using the LC-MRM method in plasma of a normal person and plasma of a macular degeneration patient using a T-test (Non AMD: non-macular degeneration patients, AMD: Macular degeneration). In the drawing, ○ means a very low or high outlier as a result of box and whisker plot analysis.
도 2는 정상인의 혈장과 당뇨망막병증 환자의 혈장에서 LC-MRM 방법을 이용하여 측정된 13개의 단백질 정량값을 T-검정으로 통계처리 하여 분석한 데이터이다 (Non DMR: 비 당뇨망막병증, DMR: 당뇨망막병증).2 is data obtained by statistically processing 13 protein quantification values measured using the LC-MRM method in plasma of normal subjects and plasma of diabetic retinopathy patients by T-test (Non DMR: non-diabetic retinopathy, DMR : Diabetic retinopathy).
도 3은 황반변성 질환(AMD)에 대표적인 마커 (A) ADAMTSL2 및 당뇨망막병증 질환(DMR)에 대표적인 마커 (B) IGFBP2의 정량값 결과를 AUC 및 T-검정으로 통계처리 하여 분석한 데이터이다. FIG. 3 is data analyzed by statistically processing results of quantification values of (A) ADAMTSL2 representative for macular degeneration disease (AMD) and (B) IGFBP2 representative marker for diabetic retinopathy (DMR) by AUC and T-test.
도 4는 단일 마커의 단백질 정량값 및 전체 황반변성 환자의 기본적인 임상정보를 결합하여 T-검정으로 통계처리 하여 분석한 데이터이다 (AMD: 나이관련 황반변성, Non AMD: 비황반변성).Figure 4 is a data obtained by combining the quantitative value of the protein of a single marker and basic clinical information of all macular degeneration patients and analyzed by statistical processing using a T-test (AMD: age-related macular degeneration, Non AMD: non-macular degeneration).
도 5는 단일 마커의 단백질 정량값 및 전체 당뇨망막병증 환자의 기본적인 임상정보를 결합하여 T-검정으로 통계처리 하여 분석한 데이터이다 (DMR: 당뇨망막병증, Non DMR: 비당뇨망막병증).5 is a data obtained by combining the quantitative value of protein of a single marker and basic clinical information of all diabetic retinopathy patients and analyzed by statistical processing using a T-test (DMR: diabetic retinopathy, Non DMR: non-diabetic retinopathy).
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명은 일관점에서, ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)으로 구성된 군에서 선택된 1종 이상의 실명 유발 안질환 진단용 마커에 관한 것이다.In a consistent aspect, the present invention is ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2). ), LGALS3BP (galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 ( Thrombospondin-1) and ZG16B (Zymogen granule protein 16 homolog B). The present invention relates to a marker for diagnosing one or more blindness-causing eye diseases selected from the group consisting of.
본 발명에서 사용된 용어 "진단"은 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 본 발명의 목적상, 진단은 실명 유발 안질환 발병 여부를 확인하는 것이다.The term "diagnosis" as used in the present invention means identifying the presence or characteristics of a pathological condition. For the purpose of the present invention, the diagnosis is to determine whether or not blindness-causing eye disease has occurred.
본 발명에서 사용된 용어 "진단용 마커"란 정상 대조군(실명 유발 안질환이 아닌 개체)에 비하여 실명 유발 안질환을 가진 개체에서 유전자 발현 수준 또는 단백질 발현 수준의 유의적인 증가 또는 감소 양상을 보이는 폴리펩티드 또는 핵산(예: mRNA 등), 지질, 당지질, 당단백질, 당(단당류, 이당류, 올리고당류 등) 등과 같은 유기 생체 분자 등을 포함한다.The term "diagnostic marker" used in the present invention refers to a polypeptide showing a significant increase or decrease in gene expression level or protein expression level in an individual with blindness-causing eye disease compared to a normal control group (individuals other than blindness-causing eye disease) or It includes organic biomolecules such as nucleic acids (eg, mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.).
본 발명에 있어서, 상기 실명 유발 안질환은 나이관련황반변성, 당뇨망막병증, 백내장 및 녹내장으로 구성된 군에서 선택된 하나 이상일 수 있다.In the present invention, the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
황반변성은 진행 정도에 따라 초기 황반변성(early AMD)와 후기 황반변성(Late AMD)로 구분한다. 초기 황반변성은 혈관이 발달하지 않으며, 후기 황반변성은 혈관이 발달하는 등 그 기전에 있어서 상이하기에, 후기 황반변성 진단용 마커로 알려진 마커라도 반드시 초기 황반변성 진단용 마커로 사용될 수는 없다. 본 발명의 마커는 초기 황반변성 및 후기 황반변성을 모두 특이적으로 진단할 수 있다.According to the degree of progression, macular degeneration is classified into early AMD and late AMD. Since early macular degeneration does not develop blood vessels, and late macular degeneration is different in its mechanisms, such as blood vessel development, even a marker known as a late macular degeneration diagnostic marker cannot necessarily be used as an early macular degeneration diagnostic marker. The markers of the present invention can specifically diagnose both early and late macular degeneration.
당뇨망막병증은 진행 정도에 따라 초기의 비증식성 당뇨망막병증(NPDR)과 후기의 증식성 당뇨망막병증(PDR)로 구분한다. 비증식성 당뇨망막병증은 혈관이 발달하지 않는 특징이 있으며, 증식성 당뇨망막병증은 혈관이 발달하는 등 그 기전에 있어서 상이하며, 비증식성 당뇨망막병증이 반드시 증식성 당뇨망막병증으로 진행되는 것이 아니어서, 증식성 당뇨망막병증의 진단용 마커로 알려진 마커라도 반드시 비증식성 당뇨망막병증의 진단용 마커로 사용될 수는 없다. 본 발명의 마커는 비증식성 당뇨망막병증 및 증식성 당뇨망막병증을 모두 특이적으로 진단할 수 있다. Diabetic retinopathy is classified into early nonproliferative diabetic retinopathy (NPDR) and late proliferative diabetic retinopathy (PDR) according to the degree of progression. Nonproliferative diabetic retinopathy is characterized by no blood vessel development, and proliferative diabetic retinopathy differs in its mechanism, such as blood vessel development, and nonproliferative diabetic retinopathy does not necessarily progress to proliferative diabetic retinopathy. Then, even a marker known as a diagnostic marker for proliferative diabetic retinopathy cannot necessarily be used as a diagnostic marker for non-proliferative diabetic retinopathy. The markers of the present invention can specifically diagnose both non-proliferative diabetic retinopathy and proliferative diabetic retinopathy.
상기 실명 유발 안질환 진단용 마커 중에서 ADAMTSL2(ADAMTS-like protein 2)는 ADAMTS(a disintegrin and metalloproteinase with thrombospondin motifs) 유사 단백질 서브 패밀리 구성원으로, 세포 표면과 세포외 기질을 바인딩하는 당단백질이다. ADAMTSL2는 LTBP1(latent transforming growth factor beta binding protein 1)과 상호작용하며, LTBP1 단백질은 세포의 성장 및 분열을 조절하는 중요한 성장인자인 TGF-β1의 저장에 관여하므로, ADAMTSL2는 TGF-β1의 이용가능성을 조절한다. Among the markers for diagnosis of blindness-causing eye diseases, ADAMTSL2 (ADAMTS-like protein 2) is a member of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-like protein subfamily, and is a glycoprotein that binds the cell surface and the extracellular matrix. ADAMTSL2 interacts with LTBP1 (latent transforming growth factor beta binding protein 1), and LTBP1 protein is involved in the storage of TGF-β1, an important growth factor that regulates cell growth and division, so ADAMTSL2 has the possibility of using TGF-β1. Adjust.
본 발명에 있어서, 상기 ADAMTSL2는 바람직하게 서열번호 1의 아미노산 서열을 포함할 수 있으나, 서열번호 1의 아미노산 서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함하는 것일 수 있다.In the present invention, the ADAMTSL2 may preferably include the amino acid sequence of SEQ ID NO: 1, but the amino acid sequence of SEQ ID NO: 1 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
Cp(Ceruloplasmin)는 혈액에서 구리를 운반하는 주단백질로서 철 대사에서 중요한 역할을 한다. 건강한 사람의 혈장에서 약 95% 이상의 구리가 세룰로플라스민(Ceruloplasmin) 형태로 존재한다. Cp (Ceruloplasmin) is the main protein that carries copper in the blood and plays an important role in iron metabolism. More than 95% of copper is present in the plasma of healthy people in the form of ceruloplasmin.
본 발명에 있어서, 상기 Cp는 바람직하게 서열번호 2의 아미노산 서열을 포함할 수 있으나, 서열번호 3의 아미노산 서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함하는 것일 수 있다.In the present invention, the Cp may preferably include the amino acid sequence of SEQ ID NO: 2, but the amino acid sequence of SEQ ID NO: 3 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
CFH(complement factor H)는 보체 활성화를 조절하여 면역반응을 유지하는데 필수적인 역할을 하는 당단백질이다. 세포표면의 같은 당사슬구조와 결합하여 보체 활성화 및 증폭을 막는 보체 억제제 역할을 한다. CFH (complement factor H) is a glycoprotein that plays an essential role in maintaining the immune response by regulating complement activation. It binds to the same sugar chain structure on the cell surface and acts as a complement inhibitor to prevent complement activation and amplification.
본 발명에 있어서, 상기 CFH는 바람직하게 서열번호 3의 아미노산 서열을 포함할 수 있으나, 서열번호 3의 아미노산 서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함하는 것일 수 있다.In the present invention, the CFH may preferably include the amino acid sequence of SEQ ID NO: 3, but the amino acid sequence of SEQ ID NO: 3 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
DDI2(Protein DDI1 homolog2)는 내부펩타이드 절단효소로써 세포 성장 및 DNA 복제 조절에 관여하는 Nrf1(nuclear respiratory factor 1)를 활성화하여 프로테아좀 이상에 의한 단백질 분해(degradation)을 보완한다. DDI2 (Protein DDI1 homolog2) is an internal peptide cleavage enzyme that activates nuclear respiratory factor 1 (Nrf1), which is involved in regulating cell growth and DNA replication, and compensates for proteasome degradation.
본 발명에 있어서, 상기 DDI2는 바람직하게 서열번호 4의 아미노산 서열을 포함할 수 있으나, 서열번호 4의 아미노산 서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함하는 것일 수 있다.In the present invention, the DDI2 may preferably include the amino acid sequence of SEQ ID NO: 4, but the amino acid sequence of SEQ ID NO: 4 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
FCN2(Ficolin 2)는 올리고렉틴의 한 종류로서 짧은 N말단 부분-콜라겐(collagen) 유사 도메인과 피브리노겐(fibrinogen) 유사 도메인으로 구성되어 있다. 주로 간에서 발현되며 박테리아 세포벽의 N-아세틸글루코사민(N-acetylglucosamin)과 결합하여, 만노오스 결합(mannose binding) 단백질처럼 옵소닌(opsonin) 역할을 함으로써 보체계의 렉틴 경로에서 중요한 역할을 한다고 알려져 있다. FCN2 (Ficolin 2) is a type of oligolectin and is composed of a short N-terminal partial-collagen-like domain and a fibrinogen-like domain. It is mainly expressed in the liver and is known to play an important role in the lectin pathway of the complement system by binding to N-acetylglucosamin in the bacterial cell wall and acting as an opsonin like a mannose binding protein.
본 발명에 있어서, 상기 FCN2는 바람직하게 서열번호 5의 아미노산 서열을 포함할 수 있으나, 서열번호 5의 아미노산 서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함하는 것일 수 있다.In the present invention, the FCN2 may preferably include the amino acid sequence of SEQ ID NO: 5, but the amino acid sequence of SEQ ID NO: 5 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
IGFBP2(insulin like growth factor binding protein 2)는 인슐린 유사생장인자(Insulin like growth factor; IGF)와 결합하여 세포 내 다양한 프로세스를 조절하며 그에 의해 혈관생성(angiogenesis)를 조절한다. 또한 다양한 암에서(전립선 및 유방암 등)에서 암세포의 생장을 촉진한다고 알려져 있다. IGFBP2 (insulin like growth factor binding protein 2) binds to insulin like growth factor (IGF) to regulate various processes within cells, thereby regulating angiogenesis. It is also known to promote the growth of cancer cells in various cancers (such as prostate and breast cancer).
본 발명에 있어서, 상기 IGFBP2는 바람직하게 서열번호 6의 아미노산 서열을 포함할 수 있으나, 서열번호 6의 아미노산 서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함하는 것일 수 있다.In the present invention, the IGFBP2 may preferably include the amino acid sequence of SEQ ID NO: 6, but the amino acid sequence of SEQ ID NO: 6 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
LGALS3BP(galectin-3-binding protein)는 LGALS3BP 유전자에 의해 암호와 되는 단백질로, Mac-2(human macrophage-associated lectin) 및 갈락틴 1(galectin 1)에 특이적으로 결합한다. LGALS3BP는 암 환자 및 HIV에 감염된 환자 혈청에서 증가하는 것으로 알려져 있으며, NK(natural killer)세포 및 LAK(lymphokine-activated killer) 세포 독성과 연관된 면역반응에 관여한다. LGALS3BP (galectin-3-binding protein) is a protein encoded by the LGALS3BP gene, and specifically binds to Mac-2 (human macrophage-associated lectin) and galactin 1 (galectin 1). LGALS3BP is known to increase in serum of cancer patients and HIV-infected patients, and is involved in immune responses associated with natural killer (NK) cells and lymphokine-activated killer (LAK) cytotoxicity.
본 발명에 있어서, 상기 LGALS3B는 바람직하게 서열번호 7의 아미노산 서열을 포함할 수 있으나, 서열번호 7의 아미노산 서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함하는 것일 수 있다.In the present invention, the LGALS3B may preferably include the amino acid sequence of SEQ ID NO: 7, but the amino acid sequence of SEQ ID NO: 7 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
MBL2(mannose-binding protein C)는 만노오스 결합 렉틴(Mannose-binding lectin; MBL) 또는 만난 결합 단백질(mannan-binding protein; MBP)으로도 불리운다. MBL2는 올리머구조(400 ~ 700 kDa)를 가지며, 대략 30kDa으로 구성된 3개의 동일한 펩타이드 사슬을 포함하는 서브 유닛으로 구성된다. 감염에 대한 반응으로 간에서 생성되며 급성 단계 단백질이라고 불리는 다른 많은 요인 중 일부에 해당한다. Mannose-binding protein C (MBL2) is also referred to as mannose-binding lectin (MBL) or mannan-binding protein (MBP). MBL2 has an oligomer structure (400-700 kDa) and is composed of subunits containing three identical peptide chains consisting of approximately 30 kDa. It is produced by the liver in response to infection and is part of a number of other factors called acute stage proteins.
본 발명에 있어서, 상기 MBL2는 바람직하게 서열번호 8의 아미노산 서열을 포함할 수 있으나, 서열번호 8의 아미노산 서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함하는 것일 수 있다.In the present invention, the MBL2 may preferably include the amino acid sequence of SEQ ID NO: 8, but the amino acid sequence of SEQ ID NO: 8 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
PNLIP(pancreatic triacylglycerol lipase)는 트리글리세라이드의 에스테르 결합을 가수분해하는 지방 분해 효소군으로, 췌장에서 분비되는 효소이다. PNLIP는 췌장 덕트 시스템을 통해 십이지장으로 분비되기 때문에 혈청 내 농도는 낮은 것으로 알려져 있으나, 췌장염이나 췌장 선암과 같은 췌장 기능이 극도로 파괴되면, PNLIP를 포함한 췌장 효소가 혈청으로 분비되기 때문에, PNLIP 혈청 농도를 측정하여 급성 췌장염을 진단할 수 있는 것으로 알려져 있다. PNLIP (pancreatic triacylglycerol lipase) is a group of lipolytic enzymes that hydrolyze the ester bond of triglycerides, and is an enzyme secreted from the pancreas. PNLIP is known to have a low serum concentration because it is secreted into the duodenum through the pancreatic duct system.However, when pancreatic functions such as pancreatitis or pancreatic adenocarcinoma are extremely destroyed, pancreatic enzymes including PNLIP are secreted into the serum. It is known that acute pancreatitis can be diagnosed by measuring.
본 발명에 있어서, 상기 PNLIP는 바람직하게 서열번호 9의 아미노산 서열을 포함할 수 있으나, 서열번호 9의 아미노산 서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함하는 것일 수 있다.In the present invention, the PNLIP may preferably include the amino acid sequence of SEQ ID NO: 9, but the amino acid sequence of SEQ ID NO: 9 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
SELE(E-selectin)는 CD62E(CD62 antigen-like family member E), ELAM-1(endothelial-leukocyte adhesion molecule 1) 또는 LECAM2(leukocyte-endothelial cell adhesion molecule 2)로 알려져 있으며, 인터류킨 1β, 종양괴사인자, 리포다당류에 의해 활성화된 혈관내피세포에 4~12시간을 정점으로 하여 일과성으로 발현, 유도하는 세포접착분자이다. SELE는 염증 조직내 혈관내피세포에 강하게 발현하고, 호중구와 단구가 혈관내피세포 위를 구르는 현상을 매개함으로써 이들 세포의 염증부위로의 침윤을 촉진한다. 암세포의 혈관내피세포와의 접착에도 관여하는 것으로 알려져 있다. SELE (E-selectin) is known as CD62E (CD62 antigen-like family member E), ELAM-1 (endothelial-leukocyte adhesion molecule 1) or LECAM2 (leukocyte-endothelial cell adhesion molecule 2), interleukin 1β, tumor necrosis factor. , It is a cell adhesion molecule that is transiently expressed and induced in vascular endothelial cells activated by lipopolysaccharides with a peak of 4 to 12 hours. SELE is strongly expressed in vascular endothelial cells in inflammatory tissues and promotes invasion of these cells into the inflammatory site by mediating the phenomenon that neutrophils and monocytes roll over vascular endothelial cells. It is known to be involved in adhesion of cancer cells to vascular endothelial cells.
본 발명에 있어서, 상기 SELE는 바람직하게 서열번호 10의 아미노산 서열을 포함할 수 있으나, 서열번호 10의 아미노산 서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함하는 것일 수 있다.In the present invention, the SELE may preferably include the amino acid sequence of SEQ ID NO: 10, but the amino acid sequence of SEQ ID NO: 10 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
SIGLEC14(Sialic acid-binding Ig-like lectin 14)는 SIGLEC(Sialic acid-binding immunoglobulin-type lectins)의 서브패밀리 중 하나로, SIGLEC는 시알산과 결합하는 세포 표면 단백질이며 주로 면역세포의 표면에서 발현된다. SIGLEC과 시알산의 단백질 상호작용은 면역 시스템을 온, 오프 시키는 스위치 역할을 하며, 암세포 역시 SIGLEC-시알산 반응을 이용하여 면역반응에 대한 저항성을 획득하는 것으로 알려져 있다. SIGLEC14 (Sialic acid-binding Ig-like lectin 14) is one of the subfamily of SIGLEC (Sialic acid-binding immunoglobulin-type lectins), and SIGLEC is a cell surface protein that binds to sialic acid and is mainly expressed on the surface of immune cells. The protein interaction between SIGLEC and sialic acid acts as a switch to turn on and off the immune system, and cancer cells are also known to acquire resistance to the immune response by using the SIGLEC-sialic acid reaction.
본 발명에 있어서, 상기 SIGLEC14는 바람직하게 서열번호 11의 아미노산 서열을 포함할 수 있으나, 서열번호 11의 아미노산 서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함하는 것일 수 있다.In the present invention, the SIGLEC14 may preferably include the amino acid sequence of SEQ ID NO: 11, but the amino acid sequence of SEQ ID NO: 11 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
THBS1(Thrombospondin-1)은 트롬보스폰딘 패밀리(thrombospondin family) 중 하나로 신혈관 생성과 종양생성을 억제하는 당단백질이다. 플라스미노겐(plasminogen), 우로키나제(urokinase), MMP, 트롬빈(thrombin) 및 카텝신(cathepsin) 등의 혈관생성과 관련 있는 프로테아제와 결합하여 내피세포의 유착, 이동, 생장을 조절한다고 알려져 있다. THBS1 (Thrombospondin-1) is one of the thrombospondin family and is a glycoprotein that inhibits neovascularization and tumorigenesis. It is known that it binds to proteases related to angiogenesis, such as plasminogen, urokinase, MMP, thrombin, and cathepsin, and regulates adhesion, migration, and growth of endothelial cells.
본 발명에 있어서, 상기 THBS1는 바람직하게 서열번호 12의 아미노산 서열을 포함할 수 있으나, 서열번호 12의 아미노산 서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함하는 것일 수 있다.In the present invention, the THBS1 may preferably include the amino acid sequence of SEQ ID NO: 12, but the amino acid sequence of SEQ ID NO: 12 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
ZG16B(Zymogen granule protein 16 homolog B)는 PAUF(pancreatic adenocarcinoma upregulated factor)로, 탄수화물과 결합하고, 내부상피세포, 혈관생성 및 침투(permeability)를 활성화 시킨다고 알려져 있다. ZG16B (Zymogen granule protein 16 homolog B) is a pancreatic adenocarcinoma upregulated factor (PAUF) that binds to carbohydrates and is known to activate internal epithelial cells, angiogenesis, and permeability.
본 발명에 있어서, 상기 ZG16B는 바람직하게 서열번호 13의 아미노산 서열을 포함할 수 있으나, 서열번호 13의 아미노산 서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함하는 것일 수 있다.In the present invention, the ZG16B may preferably include the amino acid sequence of SEQ ID NO: 13, but the amino acid sequence of SEQ ID NO: 13 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, or 99% or more of the same sequence may be included.
하지만, 상기 마커들은 나이관련황반변성, 당뇨망막병증, 백내장 또는 녹내장 등과 같은 실명 유발 안질환 진단에 사용할 수 있음을 개시한 종래 기술은 알려진 바가 없다. However, there is no known prior art that discloses that the markers can be used to diagnose blindness-causing eye diseases such as age-related macular degeneration, diabetic retinopathy, cataract or glaucoma.
본 발명은 다른 관점에서, ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)으로 구성된 군에서 선택된 1종 이상의 실명 유발 안질환 진단용 마커의 단백질 발현 수준 또는 그의 mRNA 발현 수준을 측정하는 물질을 포함하는 실명 유발 안질환 진단용 조성물에 관한 것이다. In another aspect, the present invention is ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2) ), LGALS3BP (galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 ( Thrombospondin-1) and ZG16B (Zymogen granule protein 16 homolog B), a composition for diagnosing blindness-causing eye diseases comprising a substance for measuring the protein expression level of at least one marker for diagnosis of blindness-causing eye disease or its mRNA expression level It is about.
본 발명에 있어서, 상기 실명 유발 안질환은 나이관련황반변성, 당뇨망막병증, 백내장 및 녹내장으로 구성된 군에서 선택된 하나 이상일 수 있다.In the present invention, the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
본 발명에 있어서, 상기 복합 마커의 mRNA 수준을 측정하는 제제는 상기 마커의 유전자에 특이적으로 결합하는 프라이머쌍, 프로브 또는 안티센스 뉴클레오타이드인 것을 특징으로 하며, 상기 유전자들의 핵산 정보가 GeneBank 등에 알려져 있으므로 당업자는 상기 서열을 바탕으로 이들 프라이머쌍, 프로브 또는 안티센스 뉴클레오타이드를 디자인할 수 있다.In the present invention, the agent for measuring the mRNA level of the complex marker is characterized in that it is a primer pair, probe or antisense nucleotide that specifically binds to the gene of the marker, and nucleic acid information of the genes is known in GeneBank, etc. Can design these primer pairs, probes or antisense nucleotides based on the above sequence.
본 발명에 사용된 용어 "mRNA 발현수준 측정"이란 실명 유발 안질환을 진단하기 위하여 실명 유발 안질환 의심 환자로부터 분리된 생물학적 시료에서 실명 유발 안질환 진단용 유전자들의 mRNA 존재 여부와 발현 정도를 확인하는 과정으로 mRNA의 양을 측정한다. The term "measurement of mRNA expression level" used in the present invention is a process of confirming the presence and expression of mRNA of genes for diagnosis of blindness-causing eye disease in a biological sample isolated from a patient suspected of blindness-causing eye disease in order to diagnose blindness-causing eye disease Measure the amount of mRNA.
본 발명에서 사용된 용어 "프라이머"는 표적 유전자 서열을 인지하는 단편으로서, 정방향 및 역방향의 프라이머 쌍을 포함하나, 바람직하게는, 특이성 및 민감성을 가지는 분석 결과를 제공하는 프라이머 쌍이다. 프라이머의 핵산 서열이 시료 내 존재하는 비-표적 서열과 불일치하는 서열이어서, 상보적인 프라이머 결합 부위를 함유하는 표적 유전자 서열만 증폭하고 비특이적 증폭을 유발하지 않는 프라이머일 때, 높은 특이성이 부여될 수 있다.The term "primer" as used in the present invention is a fragment that recognizes a target gene sequence, and includes forward and reverse primer pairs, preferably, a primer pair that provides an analysis result having specificity and sensitivity. When the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, a primer that amplifies only the target gene sequence containing the complementary primer binding site and does not induce non-specific amplification can give high specificity. .
본 발명에서 사용된 용어 "프로브"란 시료 내의 검출하고자 하는 표적 물질과 특이적으로 결합할 수 있는 물질을 의미하며, 상기 결합을 통하여 특이적으로 시료 내의 표적 물질의 존재를 확인할 수 있는 물질을 의미한다. 프로브의 종류는 당업계에서 통상적으로 사용되는 물질로서 제한은 없으나, 바람직하게는 PNA(peptide nucleic acid), LNA(locked nucleic acid), 펩타이드, 폴리펩타이드, 단백질, RNA 또는 DNA 일 수 있으며, 가장 바람직하게는 PNA이다. 보다 구체적으로, 상기 프로브는 바이오 물질로서 생물에서 유래되거나 이와 유사한 것 또는 생체 외에서 제조된 것을 포함하는 것으로, 예를 들어, 효소, 단백질, 항체, 미생물, 동식물 세포 및 기관, 신경세포, DNA, 및 RNA일 수 있으며, DNA는 cDNA, 게놈 DNA, 올리고뉴클레오타이드를 포함하며, RNA는 게놈 RNA, mRNA, 올리고뉴클레오타이드를 포함하며, 단백질의 예로는 항체, 항원, 효소, 펩타이드 등을 포함할 수 있다.The term "probe" used in the present invention refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in a sample through the binding. do. The type of probe is a material commonly used in the art and is not limited, but preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferred Hagi is PNA. More specifically, the probe is a biomaterial that includes an organism-derived or similar thing or a thing produced in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and It may be RNA, DNA includes cDNA, genomic DNA, oligonucleotide, RNA includes genomic RNA, mRNA, oligonucleotide, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.
본 발명에서 사용된 용어 "안티센스"는 안티센스 올리고머가 왓슨-크릭 염기쌍 형성에 의해 RNA 내의 표적 서열과 혼성화되어, 표적서열 내에서 전형적으로 mRNA와 RNA:올리고머 헤테로이중체의 형성을 허용하는, 뉴클레오티드 염기의 서열 및 서브유닛간 백본을 갖는 올리고머를 의미한다. 올리고머는 표적 서열에 대한 정확한 서열 상보성 또는 근사 상보성을 가질 수 있다.The term "antisense", as used herein, refers to a nucleotide base in which an antisense oligomer is hybridized with a target sequence in RNA by Watson-Crick base pairing, typically allowing the formation of an mRNA and RNA: oligomeric heterodimer within the target sequence. It means an oligomer having a sequence of and a backbone between subunits. Oligomers may have exact sequence complementarity or approximate complementarity to the target sequence.
본 발명에 있어서, 상기 복합 마커의 단백질 수준을 측정하는 제제는 상기 단백질 또는 펩타이드 단편에 특이적으로 결합하는 항체, 상호작용 단백질, 리간드, 나노입자(nanoparticles) 또는 압타머(aptamer)인 것을 특징으로 할 수 있다. In the present invention, the agent for measuring the protein level of the complex marker is an antibody, interacting protein, ligand, nanoparticles, or aptamer that specifically binds to the protein or peptide fragment. can do.
본 발명에 사용된 용어 "단백질 발현수준 측정"이란 실명 유발 안질환을 진단하기 위하여 생물학적 시료에서 실명 유발 안질환 진단용 마커의 유전자로부터 발현된 단백질의 존재 여부와 발현 정도를 확인하는 과정이다. The term "protein expression level measurement" used in the present invention is a process of determining the presence and expression of a protein expressed from a gene of a marker for diagnosis of blindness-causing eye disease in a biological sample in order to diagnose blindness-causing eye disease.
본 발명에 사용된 용어 "항체"는 항원과 특이적으로 결합하여 항원-항체 반응을 일으키는 물질을 가리킨다. 본 발명의 목적상, 항체는 본 발명의 당뇨망막병증 진단용 복합 바이오 마커에 대해 특이적으로 결합하는 항체를 의미한다. 본 발명의 항체는 다클론 항체, 단클론 항체 및 재조합 항체를 모두 포함한다. 상기 항체는 당업계에 널리 공지된 기술을 이용하여 용이하게 제조될 수 있다. 예를 들어, 다클론 항체는 상기 당뇨망막병증 마커 단백질 항원을 동물에 주사하고 동물로부터 채혈하여 항체를 포함하는 혈청을 수득하는 과정을 포함하는 당업계에 널리 공지된 방법에 의해 생산될 수 있다. 이러한 다클론 항체는 염소, 토끼, 양, 원숭이, 말, 돼지, 소, 개 등의 임의의 동물로부터 제조될 수 있다. 또한, 단클론 항체는 당업계에 널리 공지된 하이브리도마 방법(hybridoma method; Kohler and Milstein, European Journal of Immunology 6:511-519, 1976 참조), 또는 파지 항체 라이브러리 기술(Clackson et al, Nature, 352:624-628, 1991; Marks et al, J.
Mol
. Biol., 222:58, 1-597, 1991 참조) 을 이용하여 제조될 수 있다. 상기 방법으로 제조된 항체는 겔 전기영동, 투석, 염 침전, 이온교환 크로마토그래피, 친화성 크로마토그래피 등의 방법을 이용하여 분리, 정제될 수 있다. 또한, 본 발명의 항체는 2개의 전장의 경쇄 및 2개의 전장의 중쇄를 갖는 완전한 형태뿐만 아니라, 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적인 단편이란, 적어도 항원 결합 기능을 보유하고 있는 단편을 의미하며, Fab, F(ab'), F(ab')2및 Fv 등이 있다. 또한 본 발명의 항체는 상업적으로 입수한 것일 수 있다.As used herein, the term "antibody" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to the complex biomarker for diabetic retinopathy of the present invention. The antibodies of the present invention include all of polyclonal antibodies, monoclonal antibodies and recombinant antibodies. The antibody can be easily prepared using techniques well known in the art. For example, the polyclonal antibody can be produced by a method well known in the art, including the process of injecting the diabetic retinopathy marker protein antigen into an animal and collecting blood from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be prepared from any animal such as goat, rabbit, sheep, monkey, horse, pig, cow, dog. In addition, the monoclonal antibody is a hybridoma method well known in the art (see the hybridoma method; Kohler and Milstein, European Journal of Immunology 6:511-519, 1976), or phage antibody library technology (Clackson et al, Nature, 352 :624-628, 1991; Marks et al, J. Mol . Biol. , 222:58, 1-597, 1991). The antibody prepared by the above method may be separated and purified using a method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. In addition, the antibody of the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules. The functional fragment of an antibody molecule means a fragment that has at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2 and Fv. In addition, the antibody of the present invention may be obtained commercially.
본 발명에 사용된 용어 "PNA(Peptide Nucleic Acid)"는 인공적으로 합성된, DNA 또는 RNA와 비슷한 중합체를 가리킨다. DNA는 인산-리보스당 골격을 갖는데 반해, PNA는 펩타이드 결합에 의해 연결된 반복된 N-(2-아미노에틸)-글리신 골격을 가지며, 이로 인해 DNA 또는 RNA에 대한 결합력과 안정성이 크게 증가되어 분자 생물학, 진단 분석 및 안티센스 치료법에 사용되고 있다. PNA는 문헌(Nielsen PE et al, Science, 254(5037):1497-500, 1991)에 상세하게 개시되어 있다.The term "PNA (Peptide Nucleic Acid)" as used herein refers to an artificially synthesized, DNA or RNA-like polymer. While DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by a peptide bond, which greatly increases the binding power and stability to DNA or RNA, resulting in molecular biology. , Diagnostic analysis and antisense treatment PNA is described in detail in Nielsen PE et al, Science , 254(5037):1497-500, 1991.
본 발명에서 "앱타머"는 올리고핵산 또는 펩타이드 분자이며, 앱타머의 일반적인 내용은 문헌(Bock LC et al., Nature, 355(6360):5646, 1992; Hoppe-Seyler F and Butz K, J
Mol
Med., 78(8):42630, 2000; Cohen BA et al., Proc
Natl
Acad
Sci USA., 95(24):142727, 1998)에 상세하게 개시되어 있다.In the present invention, "aptamer" is an oligonucleotide or peptide molecule, and general information of the aptamer is described in Bock LC et al. , Nature , 355(6360):5646, 1992; Hoppe-Seyler F and Butz K, J Mol Med ., 78(8):42630, 2000; Cohen BA et al. , Proc Natl Acad Sci USA ., 95(24):142727, 1998).
본 발명은 또 다른 관점에서, 상기 실명 유발 안질환 진단용 조성물을 포함하는 실명 유발 안질환 진단용 키트에 관한 것이다. In another aspect, the present invention relates to a kit for diagnosing blindness-induced eye diseases, including the composition for diagnosing blindness-induced eye diseases.
상기 키트는 당업계에 알려져 있는 통상의 제조방법에 의해 제조될 수 있다. 상기 키트는 예를 들면, 동결 건조 형태의 항체와 완충액, 안정화제, 불활성 단백질 등을 포함할 수 있다. The kit can be prepared by a conventional manufacturing method known in the art. The kit may include, for example, an antibody in a freeze-dried form, a buffer, a stabilizer, an inactive protein, and the like.
상기 키트는 검출가능한 표지를 더 포함할 수 있다. 용어 "검출가능한 표지"는 표지가 없는 동일한 종류의 분자들 중에서 표지를 포함하는 분자를 특이적으로 검출하도록 하는 원자 또는 분자를 의미한다. 상기 검출가능한 표지는 상기 단백질 또는 그의 단편에 특이적으로 결합하는 항체, 상호작용 단백질, 리간드, 나노입자, 또는 압타머에 부착된 것일 수 있다. 상기 검출가능한 표지는 방사종(radionuclide), 형광원(fluorophore), 효소(enzyme)를 포함할 수 있다. The kit may further include a detectable label. The term "detectable label" refers to an atom or molecule that specifically detects a molecule containing a label among molecules of the same type without a label. The detectable label may be attached to an antibody, interacting protein, ligand, nanoparticle, or aptamer that specifically binds to the protein or fragment thereof. The detectable label may include a radionuclide, a fluorophore, or an enzyme.
상기 키트는 당업계에 알려진 다양한 면역분석법 또는 면역염색법에 따라 이용될 수 있다. 상기 면역분석법 또는 면역염색법은 방사능면역분석, 방사능면역침전, 면역침전, ELISA, 캡처-ELISA, 억제 또는 경쟁 분석, 샌드위치 분석, 유세포 분석, 면역형광염색 및 면역친화성 정제를 포함할 수 있다. 바람직하게, 상기 키트는 RT-PCR(reverse transcription polymerase chain reaction) 키트, DNA 칩 키트, ELISA(enzyme linked immunosorbent assay) 키트, 단백질 칩 키트, 래피드(rapid) 키트 또는 MRM(multiple reaction monitoring)인 것일 수 있다. The kit can be used according to various immunoassays or immunostaining methods known in the art. The immunoassay or immunostaining method may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA, capture-ELISA, inhibition or competition analysis, sandwich analysis, flow cytometry, immunofluorescence staining, and immunoaffinity purification. Preferably, 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. have.
또한, 상기 키트는 질량분석에 이용될 수 있다. 이 경우, 상기 단백질의 특정 아미노산 잔기는 미리스토일화(myristoylation), 이소프레닐화, 프레닐화, 글리피칸화(glypiation), 리포일화(lipoylation), 아실화, 알킬화, 메틸화, 탈메틸화, 아미드화, 유비퀴틴화, 인산화, 탈아미드화, 글리코실화, 산화, 또는 아세틸화 등과 같은 변형을 가질 수 있다. In addition, the kit can be used for mass spectrometry. In this case, the specific amino acid residues of the protein are myristoylation, isoprenylation, prenylation, glypiation, lipoylation, acylation, alkylation, methylation, demethylation, amidation, It may have modifications such as ubiquitination, phosphorylation, deamidation, glycosylation, oxidation, or acetylation.
본 발명은 또 다른 관점에서, (a) 환자의 생물학적 시료로부터 ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)으로 구성된 군에서 선택된 1종 이상의 실명 유발 안질환 진단용 마커의 mRNA 또는 단백질 수준을 측정하는 단계; 및 In another aspect of the present invention, (a) ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), from a biological sample of a patient. IGFBP2 (insulin like growth factor binding protein 2), LGALS3BP (galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding protein) Measuring the mRNA or protein level of at least one marker for diagnosis of blindness-causing eye diseases selected from the group consisting of Ig-like lectin 14), THBS1 (Thrombospondin-1) and ZG16B (Zymogen granule protein 16 homolog B); And
(b) 상기 mRNA 또는 단백질 발현 수준을 대조군 시료와 비교하는 단계를 포함하는 실명 유발 안질환 진단을 위한 정보제공 방법에 관한 것이다.(b) It relates to a method for providing information for diagnosing blindness-causing eye diseases comprising the step of comparing the mRNA or protein expression level with a control sample.
본 발명에 있어서, 상기 실명 유발 안질환은 나이관련황반변성, 당뇨망막병증, 백내장 및 녹내장으로 구성된 군에서 선택된 하나 이상일 수 있다.In the present invention, the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
상기 방법에서 "생물학적 시료(biological sample)"란 실명 유발 안질환 발병에 의해 단백질 발현 수준 또는 유전자 발현 수준이 차이가 나는 조직, 세포, 혈액, 혈청, 혈장, 타액, 뇌척수액 또는 뇨와 같은 시료 등을 의미하며, 바람직하게는 혈액, 혈장, 혈청을 의미한다. In the above method, a "biological sample" refers to a tissue, cell, blood, serum, plasma, saliva, cerebrospinal fluid, or urine with a difference in protein expression level or gene expression level due to the onset of blindness-causing eye disease. Means, and preferably means blood, plasma, and serum.
상기 실명 유발 안질환 진단을 위한 정보제공 방법은 나이, 고혈압 여부, 고지혈증 여부, 흡연 여부, 당뇨 여부, BMI (body mass index), Hb1AC 검사결과 및 심혈관 질환 여부로 구성된 군에서 선택된 1종 이상의 임상정보를 추가로 포함할 수 있다. The method of providing information for the diagnosis of blindness-causing eye disease includes at least one clinical information selected from the group consisting of age, hypertension, hyperlipidemia, smoking, diabetes, BMI (body mass index), Hb1AC test results, and cardiovascular disease. It may further include.
또한, 실명 유발 안질환 진단을 위한 정보제공 방법은 마커의 유전자 발현 수준 또는 단백질 발현 수준이 대조군에 비해 증가/감소하면 실명 유발 안질환인 것으로 판정하는 단계를 추가로 포함할 수 있다.In addition, the method of providing information for diagnosing blindness-causing eye disease may further include determining that it is a blindness-causing eye disease if the gene expression level or protein expression level of the marker increases/decreases compared to the control group.
상기 (a) 단계의 mRNA 발현 수준 측정은 상기 복합 마커의 유전자에 특이적으로 결합하는 프라이머쌍, 프로브 또는 안티센스 뉴클레오타이드를 이용하여 측정 및 비교할 수 있다.The mRNA expression level in step (a) can be measured and compared using a primer pair, a probe, or an antisense nucleotide specifically binding to the gene of the complex marker.
상기 mRNA 발현 수준 측정 또는 비교 분석 방법으로는 역전사효소 중합효소반응, 경쟁적 역전사효소 중합효소반응, 실시간 역전사효소 중합효소반응, RNase 보호 분석법, 노던 블랏팅 또는 DNA 칩 등을 사용할 수 있으나, 본 발명은 이들에 제한되는 것이 아니다. 상기 측정 방법들을 통하여 정상 대조군에서의 mRNA 발현량과 실명 유발 안질환 환자의 mRNA 발현량을 확인할 수 있고, 이들 발현량 정도를 비교함으로써 실명 유발 안질환 발병 여부를 진단 또는 예측할 수 있다.As the mRNA expression level measurement or comparative analysis method, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip, etc. can be used, but the present invention It is not limited to these. Through the above measurement methods, the mRNA expression level in the normal control group and the mRNA expression level in patients with blindness-causing eye disease can be confirmed, and the onset of blindness-induced eye disease can be diagnosed or predicted by comparing the level of these expression levels.
상기 (a) 단계의 단백질 발현 수준 측정은 단백질 또는 펩타이드 단편에 특이적으로 결합하는 항체, 상호작용 단백질, 리간드, 나노입자(nanoparticles) 또는 압타머(aptamer)를 이용하여 측정 및 비교할 수 있다.The measurement of the protein expression level in step (a) may be measured and compared using antibodies, interacting proteins, ligands, nanoparticles, or aptamers that specifically bind to proteins or peptide fragments.
단백질 발현 수준 측정 또는 비교 분석 방법으로는, 단백질 칩 분석, 면역측정법, 리간드 바인딩 어세이, MALDI-TOF(Matrix Desorption/Ionization Time of Flight Mass Spectrometry)분석, SELDITOF(Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry)분석, 방사선 면역분석, 방사 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 조직면역 염색, 보체 고정 분석법, 2차원 전기영동 분석, 액상 크로마토그래피-질량분석(liquid chromatography-Mass Spectrometry, LC-MS), LCMS/MS(liquid chromatography-Mass Spectrometry/ Mass Spectrometry), 웨스턴 블랏, 및 ELISA(enzyme linked immunosorbentassay) 등이 있으나 이로 제한되는 것은 아니다Protein expression level measurement or comparative analysis methods include protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDITOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight). Mass Spectrometry Analysis, Radiation Immunoassay, Radioimmune Diffusion Method, Okteroni Immunity Diffusion Method, Rocket Immunoelectrophoresis, Tissue Immunostaining, Complement Fixation Analysis, 2D Electrophoresis Analysis, Liquid Chromatography-Mass Spectrometry, LC-MS), LCMS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blot, and ELISA (enzyme linked immunosorbentassay), but are not limited thereto.
보다 바람직하게, 본 발명에서는 다중 반응 모니터링(multiple reaction monitoring: MRM), 병행 반응 모니터링(parallel reaction monitoring: PRM), sequential windowed data independent acquisition of the total high-resolution(SWATH), 선택 반응 모니터링(selected reaction monitoring: SRM) 또는 면역 다중 반응 모니터링(immuno multiple reaction monitoring: iMRM)을 이용하여 측정될 수도 있다.More preferably, in the present invention, multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), sequential windowed data independent acquisition of the total high-resolution (SWATH), and selected reaction monitoring (selected reaction monitoring) monitoring: SRM) or immune multiple reaction monitoring (iMRM).
MRM은 물질의 정확한 단편을 결정하여 이를 질량분석기에서 깬 후, 한 번 깨진 이온 중 특정 이온을 한 번 더 선택하여 연속적으로 연결된 검출기를 이용하여 그 수를 얻는 방법이다. MRM 방법을 이용하는 경우, 정상인 개체와 실명 유발 안질환이 의심되는 개체의 혈액 시료에서 해당 단백질 또는 그의 단편을 질량분석기를 이용하여 정량할 수 있다.MRM is a method of determining an exact fragment of a substance, breaking it in a mass spectrometer, selecting a specific ion from the broken ions once more, and obtaining the number using a detector connected in series. In the case of using the MRM method, the protein or fragment thereof can be quantified using a mass spectrometer in the blood samples of normal individuals and individuals suspected of causing blindness.
상기 (b) 단계의 mRNA 또는 단백질 발현 수준 비교는 진단의 정확도를 향상시키기 위해 통계적 방법 또는 알고리즘을 사용하여 분석할 수 있으며, 선형 또는 비선형 회귀 분석방법, 선행 또는 비선형 분류(classification) 분석방법, 로지스틱 회귀 분석방법(logistic regression), 분산분석(Analysis of Variance; ANOVA), 신경망 분석방법, 유전적 분석방법, 서포트 벡터 머신 분석방법, 계층 분석 또는 클러스터링 분석방법, 결정 트리를 이용한 계층 알고리즘 또는 커널 주성분(Kernel principal component) 분석방법, 마르코프 블랭킷(Markov Blanket) 분석방법, 회귀 특성 소거(recursive feature elimination) 또는 엔트로피-기본 회귀 특성 소거 분석방법, 전방 플로팅 서치(floating search) 또는 후방 플로팅 서치(floating search) 분석방법, 및 이들의 조합으로 구성되는 군으로부터 선택되는 분석방법을 이용할 수 있다. 본 발명에서는 바람직하게, 상기 통계적 방법은 로지스틱회귀(logistic regression) 분석 방법을 사용하였으나, 이에 제한되는 것은 아니다.The comparison of the mRNA or protein expression level in step (b) can be analyzed using a statistical method or an algorithm to improve the accuracy of diagnosis, and a linear or nonlinear regression analysis method, an advance or nonlinear classification analysis method, and logistic Logistic regression, Analysis of Variance (ANOVA), neural network analysis method, genetic analysis method, support vector machine analysis method, hierarchical analysis or clustering analysis method, hierarchical algorithm or kernel principal component using decision trees ( Kernel principal component analysis method, Markov Blanket analysis method, recursive feature elimination or entropy-basic regression feature elimination analysis method, forward floating search or rear floating search analysis An analysis method selected from the group consisting of a method, and a combination thereof may be used. In the present invention, preferably, the statistical method uses a logistic regression analysis method, but is not limited thereto.
본 발명의 구체적인 일구현예에서는, 혈장 단백체 중 나이관련황반변성 진단을 위한 바이오 마커의 정량적 검증을 위하여 184개의 혈장 시료를 분석하였으며(표 1), 당뇨망막병증 진단을 위한 바이오마커의 정량적 검증을 위하여 155개의 혈장 시료를 분석하였다 (표 2). In a specific embodiment of the present invention, 184 plasma samples were analyzed for quantitative verification of biomarkers for diagnosis of age-related macular degeneration among plasma proteins (Table 1), and quantitative verification of biomarkers for diagnosis of diabetic retinopathy was performed. For this, 155 plasma samples were analyzed (Table 2).
ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)으로 구성된 13개의 타겟 마커를 다중 반응 모니터링(multiple reaction monitoring: MRM)을 이용하여 분석하였으며, 표 3에 나타난 바와 같이, 정량분석을 위해 각 마커에 대한 펩타이드를 선별하여 SIS를 이용하여 MRM-MS 분석을 수행하였다.ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2), LGALS3BP (galectin-3) -binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin-1) and ZG16B ( 13 target markers composed of zymogen granule protein 16 homolog B) were analyzed using multiple reaction monitoring (MRM), and as shown in Table 3, peptides for each marker were selected for quantitative analysis and SIS MRM-MS analysis was performed using.
본 발명의 구체적인 다른 일구현예에서는, 단일 마커 정량에 따른 진단능을 확인하기 위해 로지스틱회귀 모델을 이용하여 단일 마커 발현량 변환정보를 입력하여 나이관련황반변성 또는 당뇨망막병증으로 분류되는 확률값을 추정하였다. In another specific embodiment of the present invention, a logistic regression model is used to input conversion information of the expression level of a single marker to confirm the diagnostic ability according to the quantification of a single marker to estimate a probability value classified as age-related macular degeneration or diabetic retinopathy. I did.
그 결과, 표 4에 나타난 바와 같이, 각 단일 마커에 대한 나이관련황반변성 진단능(AUC)은 전부 0.59 이상임을 확인하였으며, 표 5에 나타난 바와 같이, 각 단일마커에 대한 당뇨망막병증 진단능(AUC)은 전부 0.60 이상임을 확인하였다. As a result, as shown in Table 4, it was confirmed that the age-related macular degeneration diagnostic ability (AUC) for each single marker was 0.59 or higher, and as shown in Table 5, diabetic retinopathy diagnostic ability for each single marker ( AUC) was confirmed to be more than 0.60.
본 발명의 구체적인 또 다른 일구현예에서는, 단일 마커 정량결과 및 임상정보를 결합하였을 때, 진단능 향상효과를 확인하기 위해 로지스틱회귀 모델을 이용하여 바이오마커 발현량 변환정보 및 임상정보 변환수치를 입력하여 나이관련 황반변성 또는 당뇨망막병증으로 분류되는 확률값을 추정하였다. In another specific embodiment of the present invention, when a single marker quantification result and clinical information are combined, biomarker expression level conversion information and clinical information conversion value are input using a logistic regression model to confirm the effect of improving diagnostic ability. Thus, the probability of classification as age-related macular degeneration or diabetic retinopathy was estimated.
표 6에 나타난 바와 같이, 황반변성 진단능(AUC)은 전부 0.61 이상임을 확인하였으며, 표 7에 나타난 바와 같이, 당뇨망막병증 진단능(AUC)은 전부 0.71 이상으로, 단일 마커와 임상정보의 결합에 의해 실명 유발 안질환의 진단능이 향상된 것을 확인하였다. As shown in Table 6, it was confirmed that the macular degeneration diagnostic ability (AUC) was all 0.61 or higher, and as shown in Table 7, the diabetic retinopathy diagnostic ability (AUC) was all 0.71 or higher, combining a single marker and clinical information. It was confirmed that the diagnostic ability of blindness-causing eye diseases was improved by.
또한, 도 1 내지 도 5에 나타난 바와 같이, 본 발명의 마커는 실명 유발 안질환 진단에 있어서 통계적으로 유의한 수치를 보이는 것을 확인하였다.In addition, as shown in Figs. 1 to 5, it was confirmed that the markers of the present invention showed statistically significant values in diagnosing blindness-causing eye diseases.
즉, 본 발명의 13종의 마커는 실명 유발 안질환 진단에 적합한 것을 확인하였으며, 단일마커와 진단개체의 임상정보를 결합하면 실명 유발 안질환 진단능이 증가하는 것을 확인하였다.That is, it was confirmed that the 13 types of markers of the present invention are suitable for diagnosis of blindness-causing eye diseases, and it was confirmed that the diagnostic ability of blindness-causing eye diseases is increased by combining the single marker with clinical information of the diagnosis subject.
본 발명은 또 다른 관점에서, (a) ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)으로 구성된 군에서 선택된 1종 이상의 마커에 대한 mRNA 또는 단백질 발현 수준이 대조군에 비해 증가된 세포 시료에 치료제 후보물질을 처리하는 단계; 및In another aspect of the present invention, (a) ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth) factor binding protein 2), LGALS3BP (galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14) ), THBS1 (Thrombospondin-1) and ZG16B (Zymogen granule protein 16 homolog B) mRNA or protein expression level for one or more markers selected from the group consisting of increased compared to the control, the step of treating a therapeutic agent candidate ; And
(b) 상기 후보 물질 처리 후, 상기 마커의 mRNA 또는 단백질 발현 수준이 감소되었는지를 확인하는 단계를 포함하는 실명 유발 안질환 치료용 물질의 스크리닝 방법에 관한 것이다.(b) After the treatment of the candidate substance, it relates to a method for screening a substance for treating blindness-causing eye diseases, comprising the step of determining whether the mRNA or protein expression level of the marker is decreased.
본 발명에 있어서, 상기 실명 유발 안질환은 나이관련황반변성, 당뇨망막병증, 백내장 및 녹내장으로 구성된 군에서 선택된 하나 이상일 수 있다.In the present invention, the blindness-causing eye disease may be at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
상기 대조군은 실명 유발 안질환을 갖지 않는 개체로부터 유래된 세포일 수 있다.The control group may be cells derived from an individual who does not have blindness-causing eye disease.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail through examples.
이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.
실시예Example
1 : 실명 유발 안질환 환자 선정 및 혈장 채취 1: Selection of patients with eye disease causing blindness and collection of plasma
1-1 : 나이관련황반변성 환자 선정 및 혈장 채취1-1: Age-related macular degeneration patient selection and plasma collection
나이관련황반변성 환자의 혈장 시료는 분당서울대학교병원의 임상시험심사위원회의 승인을 받아 채취하였다. 혈장 단백체 이용하여 바이오마커의 정량적 검출을 위해 총 184개의 혈장 시료를 분석하였으며, 분석대상인 정상군(Non AMD) 및 나이관련황반변성(AMD) 질환군의 임상적 특징은 하기 표 1에 나타내었다.Plasma samples from patients with age-related macular degeneration were collected with the approval of the Institutional Review Board of Seoul National University Bundang Hospital. A total of 184 plasma samples were analyzed for quantitative detection of biomarkers using plasma proteins, and the clinical characteristics of the analyzed target normal group (Non AMD) and age-related macular degeneration (AMD) disease group are shown in Table 1 below.
시료 (n=184)Sample (n=184) | ||||
Non AMD(n=32)Non AMD (n=32) | AMD(n=152)AMD (n=152) | Early AMD(n=57)Early AMD (n=57) | Late AMD(n=95)Late AMD (n=95) | |
나이age | 59.3±8.159.3±8.1 | 71.2±8.371.2±8.3 | 72.1±6.772.1±6.7 | 70.7±9.170.7±9.1 |
성별(여/남)Gender (Female/Male) | 15/1715/17 | 75/7775/77 | 45/1245/12 | 30/6530/65 |
Systemic risk factorsSystemic risk factors | ||||
흡연smoking | 11 (44.4)11 (44.4) | 61 (40.1)61 (40.1) | 13 (22.8)13 (22.8) | 48 (50.5)48 (50.5) |
고혈압(%)High blood pressure(%) | 10 (31.3)10 (31.3) | 74 (48.7)74 (48.7) | 23 (40.4)23 (40.4) | 51 (53.7)51 (53.7) |
고지혈증(%)Hyperlipidemia (%) | 8 (25.0)8 (25.0) | 54 (35.5)54 (35.5) | 22 (38.6)22 (38.6) | 32 (33.7)32 (33.7) |
심혈관질환(%)Cardiovascular disease (%) | 3 (9.4)3 (9.4) | 20 (13.2)20 (13.2) | 8 (14.0)8 (14.0) | 12 (12.6)12 (12.6) |
1-2 : 당뇨망막병증 환자 선정 및 혈장 채취1-2: Diabetic retinopathy patient selection and plasma collection
당뇨망막병증 환자의 혈장 시료는 분당서울대학교병원의 임상시험심사위원회의 승인을 받아 채취하였다. 혈장 단백체 이용하여 바이오마커의 정량적 검출을 위해 총 155개의 혈장 시료를 분석하였으며, 분석대상인 정상군 (Non DMR) 및 당뇨망막병증 (DMR) 질환군의 임상적 특징은 하기 표 2에 나타내었다.Plasma samples from diabetic retinopathy patients were collected with the approval of the Institutional Review Board of Seoul National University Bundang Hospital. A total of 155 plasma samples were analyzed for quantitative detection of biomarkers using plasma proteins, and the clinical characteristics of the analyzed normal group (Non DMR) and diabetic retinopathy (DMR) disease group are shown in Table 2 below.
시료(n=155)Sample (n=155) | ||||||
Non DMR(n=36)Non DMR(n=36) | DMR(n=119)DMR (n=119) | NPDR(n=60)NPDR (n=60) | PDR(n=59)PDR (n=59) | |||
Mild(n=17)Mild (n=17) | Moderate(n=17)Moderate(n=17) | Severe(n=26)Severe(n=26) | ||||
나이age | 64.1±8.364.1±8.3 | 57.5±9.557.5±9.5 | 57.4±10.957.4±10.9 | 58.9±12.158.9±12.1 | 60.0±7.660.0±7.6 | 56.1±9.056.1±9.0 |
성별(여/남)Gender (Female/Male) | 29/729/7 | 29/9029/90 | 12/512/5 | 3/143/14 | 5/215/21 | 9/509/50 |
Hb1AcHb1Ac | 7.4±1.37.4±1.3 | 7.6±1.47.6±1.4 | 7.5±0.97.5±0.9 | 7.6±1.47.6±1.4 | 7.5±1.47.5±1.4 | 7.6±1.57.6±1.5 |
당뇨치료(%)Diabetes treatment (%) | 32(88.9)32(88.9) | 116(97.5)116(97.5) | 17(100.0)17(100.0) | 17(100.0)17(100.0) | 25(96.2)25(96.2) | 57(96.6)57(96.6) |
Systemic risk factorSystemic risk factor | ||||||
BMI(%)BMI (%) | ||||||
저 that | 4(11.1)4(11.1) | 7(5.9)7(5.9) | 0(0)0(0) | 1(5.9)1(5.9) | 3(11.5)3(11.5) | 3(5.1)3(5.1) |
정상 normal | 19(52.8)19(52.8) | 64(53.8)64(53.8) | 8(47.1)8(47.1) | 8(47.1)8(47.1) | 13(50.0)13(50.0) | 35(59.3)35(59.3) |
경도 Hardness | 12(33.3)12(33.3) | 37(31.1)37(31.1) | 8(47.1)8(47.1) | 4(23.5)4(23.5) | 9(34.6)9(34.6) | 15(25.4)15(25.4) |
고도 Altitude | 1(2.8)1(2.8) | 11(9.2)11(9.2) | 1(5.9)1(5.9) | 4(23.5)4(23.5) | 0(0)0(0) | 6(10.2)6(10.2) |
흡연(%)smoking(%) | 12(33.3)12(33.3) | 53(43.7)53(43.7) | 10(58.5)10(58.5) | 8(47.1)8(47.1) | 10(38.5)10(38.5) | 24(40.7)24(40.7) |
고혈압(%)High blood pressure(%) | 19(52.8)19(52.8) | 65(54.6)65(54.6) | 6(35.3)6(35.3) | 10(58.8)10(58.8) | 14(53.8)14(53.8) | 35(59.3)35(59.3) |
고지혈증(%)Hyperlipidemia (%) | 25(69.4)25(69.4) | 50(42.0)50(42.0) | 9(52.9)9(52.9) | 8(47.1)8(47.1) | 10(38.5)10(38.5) | 23(39.0)23(39.0) |
심혈관질환(%)Cardiovascular disease (%) | 5(13.9)5(13.9) | 9(7.6)9(7.6) | 1(5.9)1(5.9) | 1(5.9)1(5.9) | 2(7.7)2(7.7) | 5(8.5)5(8.5) |
실시예Example
2 : 2 :
펩타이드Peptide
선정 및 Selection and
MRMMRM
-MS를 이용한 -Using MS
펩타이드Peptide
분석 analysis
2-1 : 펩타이드 선정 및 합성 펩타이드 설계2-1: Peptide selection and synthetic peptide design
본 발명에서는 실명 유발 안질환을 진단하기 위한 바이오 마커로 ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)의 13개를 선별하였다.In the present invention, as biomarkers for diagnosing blindness-causing eye diseases, ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 ( insulin like growth factor binding protein 2), LGALS3BP (galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-) like lectin 14), THBS1 (Thrombospondin-1), and ZG16B (Zymogen granule protein 16 homolog B) were selected.
상기 바이오마커에 대한 MRM 분석을 수행하기 위해 13 개의 바이오마커의 단백질에 대해 특정적인 전하 대 질량비(m/z)를 갖는 대표 펩타이드를 선정하고(Q1), 이 펩타이드를 전기적인 충격으로 깨서 발생하는 단편화 이온(fragmentation ion)중 가장 강도가 높은 이온(Q3)를 선정하였다. In order to perform MRM analysis on the biomarker, a representative peptide having a specific charge-to-mass ratio (m/z) for 13 biomarker proteins is selected (Q1), and the peptide is broken by electric shock. Among the fragmentation ions, the ion (Q3) having the highest strength was selected.
단백질 당 감도 높은 1개 이상의 펩티드를 측정하고 이를 기초로 질량분석기에 주입하여 각 트랜지션 당 파편화 에너지 최적값을 얻고, 강도를 기준으로 3개 이상의 상위 단편화 이온을 선택하였다 (표 3). At least one peptide with high sensitivity per protein was measured and injected into a mass spectrometer based on this to obtain an optimal value of fragmentation energy per transition, and three or more upper fragmented ions were selected based on the intensity (Table 3).
유전자명Gene name | 트립신절편Trypsin fragment | 서열번호Sequence number | 표적이온Target ion | 표적 m/zTarget m/z |
ADAMTSL2ADAMTSL2 | NFNIAGTVVK NFNIAGTVVK | 서열번호 14SEQ ID NO: 14 | +2y6+2y6 | 531.801/574.356531.801/574.356 |
CpCp | GEFYIGSK GEFYIGSK | 서열번호 15SEQ ID NO: 15 | +2y6+2y6 | 450.728/714.382450.728/714.382 |
AEVGDTI AEVGDTI | 서열번호 16SEQ ID NO: 16 | +2y5+2y5 | 430.727/561.299430.727/561.299 | |
CFHCFH | SLGNIIMVCR SLGNIIMVCR | 서열번호 17SEQ ID NO: 17 | +2y5+2y5 | 581.807/678.343581.807/678.343 |
SLGNVIMVCR SLGNVIMVCR | 서열번호 18SEQ ID NO: 18 | +2y5+2y5 | 574.799/678.343574.799/678.343 | |
CYFPYLENGYNQNYGR CYFPYLENGYNQNYGR | 서열번호 19SEQ ID NO: 19 | +2y14+2+2y14+2 | 1029.444/867.8971029.444/867.897 | |
CYFPYLENGYNQNHGR CYFPYLENGYNQNHGR | 서열번호 20SEQ ID NO: 20 | +3y13+2+3y13+2 | 677.964/781.361677.964/781.361 | |
DDI2DDI2 | DGDVVILR DGDVVILR | 서열번호 21SEQ ID NO: 21 | +2y4+2y4 | 443.753/500.355443.753/500.355 |
IDFSSIAVPGTSSPR IDFSSIAVPGTSSPR | 서열번호 22SEQ ID NO: 22 | +2y7+2y7 | 767.399/701.358767.399/701.358 | |
FCN2FCN2 | LQAADTCPEVK LQAADTCPEVK | 서열번호 23SEQ ID NO: 23 | +2y8+2y8 | 616.303/919.419616.303/919.419 |
IGFBP2IGFBP2 | LIQGAPTIR LIQGAPTIR | 서열번호 24SEQ ID NO: 24 | +2y6+2y6 | 484.798/614.362484.798/614.362 |
LGALS3BPLGALS3BP | SDLAVPSELALLK SDLAVPSELALLK | 서열번호 25SEQ ID NO: 25 | +2y8+2y8 | 678.393/870.529678.393/870.529 |
MBL2MBL2 | WLTFSLGK WLTFSLGK | 서열번호 26SEQ ID NO: 26 | +2y6+2y6 | 476.269/652.366476.269/652.366 |
PNLIPPNLIP | TGYTQASQNIR TGYTQASQNIR | 서열번호 27SEQ ID NO: 27 | +2y6+2y6 | 619.81/688.374619.81/688.374 |
GEENWLANVCK GEENWLANVCK | 서열번호 28SEQ ID NO: 28 | +2y5+2y5 | 660.306/591.292660.306/591.292 | |
VTGHILVSLFGNK VTGHILVSLFGNK | 서열번호 29SEQ ID NO: 29 | +3y4+3y4 | 462.270/465.246462.270/465.246 | |
SELESELE | NWAPGEPNNR NWAPGEPNNR | 서열번호 30SEQ ID NO: 30 | +2y7+2y7 | 577.771/783.374577.771/783.374 |
QPQNGSVRQPQNGSVR | 서열번호 31SEQ ID NO: 31 | +2y6+2y6 | 443.230/660.342443.230/660.342 | |
SIGLEC14SIGLEC14 | EGGEFTCREGGEFTCR | 서열번호 32SEQ ID NO: 32 | +2y3+2y3 | 478.201/436.197478.201/436.197 |
THBS1THBS1 | GGVNDNFQGVLQNVRGGVNDNFQGVLQNVR | 서열번호 33SEQ ID NO: 33 | +2y7+2y7 | 808.911/785.463808.911/785.463 |
ZG16BZG16B | YFSTTEDYDHEITGLR YFSTTEDYDHEITGLR | 서열번호 34SEQ ID NO: 34 | +3y7+3y7 | 649.63/825.458649.63/825.458 |
정량성을 확인하기 위하여 13개의 단백질에 해당하는 SIS(Stable-isotope labeled standard) 펩타이드를 이용하였으며, 칼리브레이션을 통하여 선형성을 확인하는 실험을 이용하여 spiked heavy peptide 농도를 정하였다. SIS 펩타이드는 펩타이드 C-말단의 라이신(Lys, K)이나 아르기닌(Arg, R) 아미노산에 있는 12C와 14N을 13C와 15N으로 치환한 펩타이드이다. 이는 혈액 내에 존재하는 내인성 펩타이드(endogenous peptide)와는 질량 값이 차이가 나지만, 동일한 서열을 갖기 때문에 펩타이드 소수성(hydrophobicity)이 동일하므로, 크로마토그램 상에서 동일한 머무름 시간(retention time, RT)에 용출된다.In order to confirm the quantification, SIS (Stable-isotope labeled standard) peptides corresponding to 13 proteins were used, and the spiked heavy peptide concentration was determined using an experiment to confirm linearity through calibration. SIS peptide is a peptide obtained by substituting 13C and 15N for 12C and 14N in the amino acids of lysine (Lys, K) or arginine (Arg, R) at the C-terminus of the peptide. This has a difference in mass value from the endogenous peptide present in blood, but since it has the same sequence, the peptide hydrophobicity is the same, so it is eluted at the same retention time (RT) on the chromatogram.
2-2 : 혈장시료의 전처리 및 MRM-MS 분석2-2: Plasma sample pretreatment and MRM-MS analysis
상기 실시예 1에서 수득한 각 혈장을 그대로 사용하거나, 더 정확한 단백질 정량을 위하여 고량(high abundant)으로 존재하는 단백질 14종(Albumin, IgG, Antitrypsin, IgA, Transferrin, Haptoglobin, Fibrinogen, Alpha2-Macroglobulin, Alpha1-Acid glycoprotein, IgM, Apolipoprotein AI, Apolipoprotein AII, Complement C3, Transthyretin)을 제거하는 감손과정(depletion)을 실시하였다. 감손은 MARS(Multiple affinity removal system, Agilent, 미국) 컬럼을 제조사의 방법대로 이용하여 14종의 단백질을 제거하고, 나머지 소량으로 존재하는 단백질을 용출시켜 분석에 사용하였다. 이 용출된 감손시료에 8 M이 되도록 요소(urea)를 첨가한 후 80 mM이 되도록 2-카르보실에틸트리스포스파인(Tris(2-carboxyethyl)-phosphine, TECP) 및 320 mM이 되도록 2-클로로아세타마이드(2-chloroacetamide)을 첨가하고 25℃에서 1시간 동안 반응시켜 이황화결합을 환원 및 알킬화를 하였다. 여기에 혈장 단백질과 라이스씨(wako) 효소의 질량 비율이 100:1이 되도록 라이스씨 효소를 넣고 실온에서 4시간 동안 반응시켰다. 그 후, 50 mM 트리스(Tris, pH8.0) 용액으로 10배 희석하고 시퀀싱이 가능한 수준의 트립신(Promega)을 단백질과 트립신의 질량 비율이 50:1이 되도록 첨가하여 37℃에서 12시간 이상 반응시켜 펩티드 절편으로 분해하였다. 0.3%가 되도록 포름산(formic acid) 용액을 가하여 pH 2.0 이하로 산성화시키고 트립신 반응을 멈추게 하였다. Each plasma obtained in Example 1 was used as it is, or 14 kinds of proteins (Albumin, IgG, Antitrypsin, IgA, Transferrin, Haptoglobin, Fibrinogen, Alpha2-Macroglobulin, etc.) exist in high amounts for more accurate protein quantification. Alpha1-Acid glycoprotein, IgM, Apolipoprotein AI, Apolipoprotein AII, Complement C3, Transthyretin) were removed. For the depletion, 14 kinds of proteins were removed using a MARS (Multiple affinity removal system, Agilent, USA) column according to the manufacturer's method, and the remaining proteins were eluted and used for analysis. After adding urea to a concentration of 8 M to the eluted depleted sample, 2-carboxylethyltrisphosphine (Tris(2-carboxyethyl)-phosphine, TECP) to be 80 mM, and 2-chloro to be 320 mM. Acetamide (2-chloroacetamide) was added and reacted at 25° C. for 1 hour to reduce and alkylate disulfide bonds. Rice seed enzyme was added so that the mass ratio of plasma protein to rice seed (wako) enzyme was 100:1, and reacted at room temperature for 4 hours. After that, dilute 10 times with 50 mM Tris (Tris, pH 8.0) solution and add sequencing trypsin (Promega) so that the mass ratio of protein and trypsin becomes 50:1, and react at 37°C for 12 hours or more. And digested into peptide fragments. Formic acid (formic acid) solution was added to a concentration of 0.3%, acidified to a pH of 2.0 or less, and the trypsin reaction was stopped.
여기에 탈염화를 진행하고 상기 실시예 2-1에서 정한 내부 표준 물질(SIS 펩타이드)로 heavy-labeled 펩타이드를 첨가(spiking)하여 MRM 분석을 시행했다. Nano ultra 2D plus(Eksigent)에 결합되어 있고, 삼중 사극자 질량분석기인 QTarp 5500(SCIEX) 장비를 이용하여 각 선정 단백질의 트랜지션에 대한 scheduled MRM 모드로 모니터링 하였다.Here, desalination was performed and a heavy-labeled peptide was added (spiking) as an internal standard material (SIS peptide) determined in Example 2-1 to perform MRM analysis. Nano ultra 2D plus (Eksigent), a triple quadrupole mass spectrometer, QTarp 5500 (SCIEX) was used to monitor the transition of each selected protein in a scheduled MRM mode.
구체적으로, 5500 볼트의 이온 전압을 사용하고 및 Q1(Quadruple 1)과 Q3(Quadruple 3)에서의 해상능을 유닛(unit)으로 설정하였다. 트랜지션에 총 사이클 시간이 1.5초가 되도록 설정하였다. 20 유닛(unit)의 분무 가스(nebulizing gas)를 사용하고 히터 온도를 350℃로 설정하였다. 배치(batch) 간 변이를 보정하기 위해 각 시료에 내부 표준 물질을 스파이킹 하고 동시에 모니터링 하였다. Specifically, an ion voltage of 5500 volts was used, and the resolution at Q1 (Quadruple 1) and Q3 (Quadruple 3) was set as a unit. The total cycle time for the transition was set to be 1.5 seconds. 20 units of nebulizing gas were used and the heater temperature was set to 350°C. Internal standards were spiked into each sample and monitored simultaneously to correct for variations between batches.
2-3 : 데이터 분석2-3: data analysis
스카이라인(Skyline; McCoss lab, University of Washington, 미국)를 이용하여 로우 데이터(raw data)를 프로세싱하여 트랜지션의 피크면적을 계산하였다. 내부표준물질 펩타이드(endogenous/heavy labeled peptide)에 대하여 피크면적을 사용하여 상대적 농도를 비교하였다. 이렇게 측정된 결과값을 이용하여 각 단백질 펩타이드의 질환 예측력(predictive ability)를 측정하고자 T-검정(T-test) 및 AUROC(Area under the receiver operating characteristic) 수치를 생성하였으며, 복합 바이오 마커군 결과와 임상정보가 결합된 예측력을 확인하고자, 로지스틱 회귀분석(logistic regression) 모델을 이용하여 상기 발현량 변환정보 및 상기 표 1 또는 표 2의 임상정보 변환정도를 입력하여 실명 유발 안질환으로 분류되는 확률값을 추정하였다. MedCal ver 17.1(MedCalc)를 사용하여 모든 통계 분석을 수행하였다.The peak area of the transition was calculated by processing raw data using Skyline (Mccoss lab, University of Washington, USA). Relative concentrations were compared for the endogenous/heavy labeled peptide using the peak area. Using the measured results, T-test and Area under the receiver operating characteristic (AUROC) values were generated to measure the predictive ability of each protein peptide. In order to confirm the predictive power combined with clinical information, the expression level conversion information and the degree of conversion of the clinical information in Table 1 or 2 are input using a logistic regression model to determine the probability value classified as a blindness-causing eye disease. Estimated. All statistical analyzes were performed using MedCal ver 17.1 (MedCalc).
상기 임상정보에는 문진을 통하여 확보한 키와 체중에 의해 계산된 신체질량지수(Body Mass Index, BMI), 흡연유무, 고지혈증 유무, 고혈압 유무, 심혈관 질환 유무, 당화혈색소(Hb1Ac) 수치를 반영하였다. 이 중 유무를 사용하는 정보는 유=1, 무=0(금연=0.5)로 변환하여 반영하였다.In the clinical information, the body mass index (BMI), smoking, hyperlipidemia, hypertension, cardiovascular disease, and glycated hemoglobin (Hb1Ac) values were reflected in the clinical information. Among them, the information using the presence or absence was converted into existence = 1, nothing = 0 (no smoking = 0.5) and reflected.
실시예Example
3 : 실명 유발 안질환 진단용 3: For diagnosis of blindness-causing eye diseases
마커Marker
분석 결과에 따른 According to the analysis result
진단능Diagnostic ability
확인 Confirm
3-1 : 나이관련 황반변성 진단능 확인3-1: Confirmation of age-related macular degeneration diagnostic ability
본 발명에서는 발굴한 혈액 마커 진단능을 확인하기 위해, 상기 실시예 2에서 측정된 바이오마커 발현량 변환정보를 입력하여 나이관련황반변성으로 분류되는 확률값을 측정하였다.In the present invention, in order to confirm the diagnostic ability of the discovered blood markers, the biomarker expression level conversion information measured in Example 2 was input to measure a probability value classified as age-related macular degeneration.
1One | 22 | 33 | 44 | 55 | 66 | 77 | 88 | 99 | 1010 | 1111 | 1212 | 1313 | |
AUCAUC | 0.710.71 | 0.650.65 | 0.610.61 | 0.610.61 | 0.600.60 | 0.680.68 | 0.660.66 | 0.610.61 | 0.610.61 | 0.630.63 | 0.600.60 | 0.600.60 | 0.600.60 |
1. ADAMTSL2, 2. CFH, 3. CP, 4, DDI2, 5. FCN2, 6. IGFBP2. 7. LGALS3BP, 8. MBL2, 9. PNLIP, 10. SELC, 11. SIGLEC14, 12. THBS1, 13. ZG16B1. ADAMTSL2, 2. CFH, 3. CP, 4, DDI2, 5. FCN2, 6. IGFBP2. 7. LGALS3BP, 8. MBL2, 9. PNLIP, 10. SELC, 11. SIGLEC14, 12. THBS1, 13. ZG16B
표 4에서 나타난 바와 같이, 각 단일 마커에 대한 나이관련황반변성 진단능(AUC)은 전부 0.6 이상임을 확인하였다. 도 1은 13개의 마커 단백질 정량값에 대한 독립적인 T-검정결과를 나타낸 것으로, 통계적으로 유의한 수치를 보이는 것을 확인하였다.As shown in Table 4, it was confirmed that the diagnostic ability (AUC) of age-related macular degeneration for each single marker was 0.6 or higher. 1 shows the independent T-test results for quantification values of 13 marker proteins, and it was confirmed that statistically significant values were shown.
3-2 : 당뇨망막병증 진단능 확인3-2: Diabetic retinopathy diagnosis ability check
본 발명에서는 발굴한 혈액 마커 진단능을 확인하기 위해, 상기 실시예 2에서 측정된 바이오마커 발현량 변환정보를 입력하여 당뇨망막병증으로 분류되는 확률값을 측정하였다.In the present invention, in order to confirm the diagnostic ability of the discovered blood marker, the biomarker expression level conversion information measured in Example 2 was input to measure a probability value classified as diabetic retinopathy.
1One | 22 | 33 | 44 | 55 | 66 | 77 | 88 | 99 | 1010 | 1111 | 1212 | 1313 | |
AUCAUC | 0.600.60 | 0.670.67 | 0.580.58 | 0.570.57 | 0.630.63 | 0.630.63 | 0.670.67 | 0.670.67 | 0.610.61 | 0.580.58 | 0.640.64 | 0.580.58 | 0.580.58 |
1. ADAMTSL2, 2. CFH, 3. CP, 4, DDI2, 5. FCN2, 6. IGFBP2. 7. LGALS3BP, 8. MBL2, 9. PNLIP, 10. SELC, 11. SIGLEC14, 12. THBS1, 13. ZG16B1. ADAMTSL2, 2. CFH, 3. CP, 4, DDI2, 5. FCN2, 6. IGFBP2. 7. LGALS3BP, 8. MBL2, 9. PNLIP, 10. SELC, 11. SIGLEC14, 12. THBS1, 13. ZG16B
표 5에 나타난 바와 같이, 각 단일마커에 대한 당뇨망막병증 진단능(AUC)은 전부 0.58 이상임을 확인하였다. 도 2는 13개의 마커 단백질 정량값에 대한 독립적인 t-검정결과를 나타낸 것으로, 통계적으로 유의한 수치를 보이는 것을 확인하였다.또한, 황반변성 및 당뇨망막병증에서 높은 진단능을 보인 ADAMTSL2(황반변성) 및 IGFBP2(당뇨망막병증)에 대한 정량값 결과를 AUC 및 T-검정으로 통계처리 하여 분석한 결과, 도 3에 나타난 바와 같이, 통계적으로 유의한 수치를 보이는 것을 확인하였다. 이는 본 발명의 마커는 실명 유발 안질환의 진단에 적합하다는 것을 의미한다.As shown in Table 5, it was confirmed that all diabetic retinopathy diagnostic ability (AUC) for each single marker was 0.58 or higher. Figure 2 shows the results of independent t-tests for quantification values of 13 marker proteins, and it was confirmed that statistically significant values were shown. In addition, ADAMTSL2 (macular degeneration), which showed high diagnostic ability in macular degeneration and diabetic retinopathy ) And IGFBP2 (diabetic retinopathy) was analyzed by statistically processing the results of the quantitative value for the AUC and T-test, as shown in Figure 3, it was confirmed to show a statistically significant value. This means that the markers of the present invention are suitable for diagnosis of blindness-causing eye diseases.
실시예Example
4 : 실명 유발 안질환 진단용 4: For diagnosis of blindness-causing eye diseases
마커Marker
분석 결과 및 임상정보 결합에 따른 진단능 확인 Confirmation of diagnostic ability according to the combination of analysis results and clinical information
4-1 : 나이관련 황반변성 진단능 확인4-1: Confirmation of age-related macular degeneration diagnostic ability
본 발명에서는 발굴한 혈액 마커 결과 및 표 1의 임상정보를 결합하였을 때 진단능을 확인하기 위해, 로지스틱회귀 모델을 이용하여 바이오마커 발현량 변환정보 및 임상정보 변환정도를 입력하여 나이관련황반변성으로 분류되는 확률값을 측정하였다.In the present invention, in order to confirm the diagnostic ability when the discovered blood marker results and the clinical information in Table 1 are combined, the conversion information of the amount of biomarker expression and the degree of conversion of the clinical information are input using a logistic regression model, The probability value to be classified was measured.
1One | 22 | 33 | 44 | 55 | 66 | 77 | 88 | 99 | 1010 | 1111 | 1212 | 1313 | |
AUCAUC | 0.720.72 | 0.620.62 | 0.620.62 | 0.620.62 | 0.610.61 | 0.690.69 | 0.660.66 | 0.620.62 | 0.620.62 | 0.620.62 | 0.620.62 | 0.610.61 | 0.610.61 |
1. ADAMTSL2, 2. CFH, 3. CP, 4, DDI2, 5. FCN2, 6. IGFBP2. 7. LGALS3BP, 8. MBL2, 9. PNLIP, 10. SELC, 11. SIGLEC14, 12. THBS1, 13. ZG16B1. ADAMTSL2, 2. CFH, 3. CP, 4, DDI2, 5. FCN2, 6. IGFBP2. 7. LGALS3BP, 8. MBL2, 9. PNLIP, 10. SELC, 11. SIGLEC14, 12. THBS1, 13. ZG16B
표 6에서 나타난 바와 같이, 나이관련황반변성 진단능(AUC)은 전부 0.61 이상임을 확인하였다. 도 4는 단일 마커 단백질 정량값 및 임상정보를 이용한 독립적인 T-검정결과를 나타낸 것으로, 단일 마커 단백질 정량값 및 임상정보의 결합이 통계적으로 유의한 수치를 보이는 것을 확인하였다.As shown in Table 6, it was confirmed that all age-related macular degeneration diagnostic ability (AUC) were 0.61 or higher. 4 shows the independent T-test results using a single marker protein quantification value and clinical information, and it was confirmed that the combination of a single marker protein quantification value and clinical information showed statistically significant values.
4-2 : 당뇨망막병증 진단능 확인4-2: Diabetic retinopathy diagnosis ability check
본 발명에서는 발굴한 혈액 마커 결과 및 표 2의 임상정보를 결합하였을 때 진단능을 확인하기 위해, 로지스틱회귀 모델을 이용하여 바이오마커 발현량 변환정보 및 임상정보 변환정도를 입력하여 당뇨망막병증으로 분류되는 확률값을 측정하였다.In the present invention, in order to confirm the diagnostic ability when the discovered blood marker results and the clinical information in Table 2 are combined, the biomarker expression level conversion information and the clinical information conversion level are input using a logistic regression model and classified as diabetic retinopathy. The probability value to be obtained was measured.
1One | 22 | 33 | 44 | 55 | 66 | 77 | 88 | 99 | 1010 | 1111 | 1212 | 1313 | |
AUCAUC | 0.710.71 | 0.760.76 | 0.710.71 | 0.710.71 | 0.720.72 | 0.750.75 | 0.710.71 | 0.740.74 | 0.730.73 | 0.710.71 | 0.730.73 | 0.710.71 | 0.710.71 |
1. ADAMTSL2, 2. CFH, 3. CP, 4, DDI2, 5. FCN2, 6. IGFBP2. 7. LGALS3BP, 8. MBL2, 9. PNLIP, 10. SELC, 11. SIGLEC14, 12. THBS1, 13. ZG16B1. ADAMTSL2, 2. CFH, 3. CP, 4, DDI2, 5. FCN2, 6. IGFBP2. 7. LGALS3BP, 8. MBL2, 9. PNLIP, 10. SELC, 11. SIGLEC14, 12. THBS1, 13. ZG16B
표 7에 나타난 바와 같이, 당뇨망막병증 진단능(AUC)은 전부 0.71 이상임을 확인하였다. 도 5는 단일 마커 단백질 정량값 및 임상정보를 이용한 독립적인 T-검정결과를 나타낸 것으로, 단일 마커 단백질 정량값 및 임상정보의 결합이 통계적으로 유의한 수치를 보이는 것을 확인하였다.As shown in Table 7, it was confirmed that all diabetic retinopathy diagnostic ability (AUC) was 0.71 or higher. 5 shows the independent T-test results using a single marker protein quantification value and clinical information, and it was confirmed that the combination of a single marker protein quantification value and clinical information showed a statistically significant value.
본 발명에서 제공하는 실명 유발 안질환 진단용 마커, 진단용 조성물, 진단용 키트 또는 진단에 필요한 정보를 제공하는 방법은 환자의 혈장을 이용하는 새로운 면역학적 진단 도구로서, 민감도가 우수할 뿐만 아니라 생검을 이용하지 않고 혈장을 대상으로 간편하게 분석할 수 있을 뿐만 아니라, 진단용 마커의 단백질 정량값과 기본적인 임상정보를 결합하여 분석하면 높은 진단능을 보이는 것을 확인하였으므로, 실명 유발 안질환의 진단에 유용하게 사용될 수 있다.The method for diagnosing blindness-causing eye diseases provided by the present invention, a diagnostic composition, a diagnostic kit, or a method for providing information necessary for diagnosis is a new immunological diagnostic tool using plasma of a patient, and has excellent sensitivity and does not use a biopsy. In addition to being able to conveniently analyze plasma as a target, it has been confirmed that high diagnostic ability is shown by combining the quantitative value of the protein and basic clinical information of the diagnostic marker, and thus it can be usefully used for diagnosis of blindness-causing eye diseases.
Claims (12)
- ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)으로 구성된 군에서 선택된 1종 이상의 실명 유발 안질환 진단용 마커.ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2), LGALS3BP (galectin-3) -binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin-1) and ZG16B ( Zymogen granule protein 16 homolog B) A marker for diagnosis of one or more blindness-causing eye diseases selected from the group consisting of.
- 제1항에 있어서, 상기 실명 유발 안질환은 나이관련황반변성, 당뇨망막병증, 백내장 및 녹내장으로 구성된 군에서 선택된 하나 이상인 것을 특징으로 하는 실명 유발 안질환 진단용 마커.The marker for diagnosing blindness-induced eye disease according to claim 1, wherein the blindness-causing eye disease is at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
- ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)으로 구성된 군에서 선택된 1종 이상의 실명 유발 안질환 진단용 마커의 단백질 수준 또는 그의 mRNA 수준을 측정하는 물질을 포함하는 실명 유발 안질환 진단용 조성물.ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2), LGALS3BP (galectin-3) -binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin-1) and ZG16B ( Zymogen granule protein 16 homolog B) A composition for diagnosing blindness-causing eye diseases comprising a substance for measuring the protein level or mRNA level of at least one marker for diagnosis of blindness-causing eye disease selected from the group consisting of.
- 제3항에 있어서, 상기 실명 유발 안질환은 나이관련황반변성, 당뇨망막병증, 백내장 및 녹내장으로 구성된 군에서 선택된 하나 이상인 것을 특징으로 하는 실명 유발 안질환 진단용 조성물.The composition for diagnosing blindness-induced eye disease according to claim 3, wherein the blindness-induced eye disease is at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, cataracts, and glaucoma.
- 제3항에 있어서, 상기 복합 마커의 mRNA 수준을 측정하는 물질은 상기 마커의 유전자에 특이적으로 결합하는 프라이머쌍, 프로브 또는 안티센스 뉴클레오타이드인 것을 특징으로 하는 실명 유발 안질환 진단용 조성물.The composition for diagnosing blindness-induced eye disease according to claim 3, wherein the substance for measuring the mRNA level of the complex marker is a primer pair, a probe, or an antisense nucleotide that specifically binds to the gene of the marker.
- 제3항에 있어서, 상기 복합 마커의 단백질 수준을 측정하는 물질은 상기 마커의 단백질 또는 펩타이드 단편에 특이적으로 결합하는 항체, 상호작용 단백질, 리간드, 나노입자(nanoparticles) 또는 압타머(aptamer)인 것을 특징으로 하는 실명 유발 안질환 진단용 조성물.The method of claim 3, wherein the substance measuring the protein level of the complex marker is an antibody, interacting protein, ligand, nanoparticles, or aptamer that specifically binds to the protein or peptide fragment of the marker. A composition for diagnosing blindness-induced eye diseases, characterized in that.
- 제3항 내지 제6항 중 어느 한 항의 실명 유발 안질환 진단용 조성물을 포함하는 실명 유발 안질환 진단용 키트.A kit for diagnosing blindness-induced eye disease, comprising the composition for diagnosing blindness-induced eye disease according to any one of claims 3 to 6.
- 제7항에 있어서, 상기 키트는 RT-PCR(Reverse transcription polymerase chain reaction) 키트, DNA 칩 키트, ELISA (Enzymelinked immunosorbent assay) 키트, 단백질 칩 키트, 래피드(rapid) 키트 또는 MRM(Multiple reaction monitoring) 키트인 것을 특징으로 하는 실명 유발 안질환 진단용 키트.The method of claim 7, wherein the kit is RT-PCR (Reverse transcription polymerase chain reaction) kit, DNA chip kit, ELISA (Enzymelinked immunosorbent assay) kit, protein chip kit, rapid kit, or MRM (Multiple reaction monitoring) kit. A kit for diagnosing blindness-induced eye diseases, characterized in that.
- (a) 환자의 생물학적 시료로부터 ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)으로 구성된 군에서 선택된 1종 이상의 실명 유발 안질환 진단용 마커의 mRNA 또는 단백질 수준을 측정하는 단계; 및 (a) ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein) from biological samples from patients 2), LGALS3BP (galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin-1) and ZG16B (Zymogen granule protein 16 homolog B) measuring the mRNA or protein level of at least one marker for diagnosis of blindness-causing eye disease selected from the group consisting of; And(b) 상기 mRNA 또는 단백질 발현 수준을 대조군 시료와 비교하는 단계를 포함하는 실명 유발 안질환 진단을 위한 정보제공 방법.(b) a method of providing information for diagnosing blindness-causing eye diseases comprising the step of comparing the mRNA or protein expression level with a control sample.
- 제9항에 있어서, 상기 실명 유발 안질환 진단을 위한 정보제공 방법은 환자의 나이, BMI(body mass index), Hb1Ac 검사결과, 고혈압 여부, 고지혈증 여부 및 심혈관 질환 여부로 구성된 군에서 선택된 1종 이상의 임상정보를 추가로 제공하는 것을 특징으로 하는 실명 유발 안질환 진단을 위한 정보제공 방법.The method of claim 9, wherein the method of providing information for diagnosing blindness-causing eye diseases is at least one selected from the group consisting of the patient's age, body mass index (BMI), Hb1Ac test result, high blood pressure, hyperlipidemia, and cardiovascular disease. Information providing method for diagnosing blindness-causing eye diseases, characterized in that providing additional clinical information.
- 제9항에 있어서, 상기 실명 유발 안질환 진단을 위한 정보제공 방법은 (c) 실명 유발 안질환 진단용 마커의 유전자 발현 수준 또는 단백질 발현 수준이 대조군에 비해 증가하면 실명 유발 안질환이라고 판정하는 단계를 추가로 포함하는 것을 특징으로 하는 실명 유발 안질환 진단을 위한 정보제공 방법.The method of claim 9, wherein the method of providing information for diagnosing blindness-causing eye disease comprises: (c) determining that the gene expression level or protein expression level of the diagnostic marker for blindness-causing eye disease is increased compared to the control group, A method of providing information for diagnosing blindness-causing eye diseases, comprising:
- (a) ADAMTSL2(ADAMTS-like protein 2), Cp(Ceruloplasmin), CFH(complement factor H), DDI2(Protein DDI1 homolog2), FCN2(Ficolin 2), IGFBP2(insulin like growth factor binding protein 2), LGALS3BP(galectin-3-binding protein), MBL2(mannose-binding protein C), PNLIP(pancreatic triacylglycerol lipase), SELE(E-selectin), SIGLEC14(Sialic acid-binding Ig-like lectin 14), THBS1(Thrombospondin-1) 및 ZG16B(Zymogen granule protein 16 homolog B)으로 구성된 군에서 선택된 1종 이상의 마커에 대한 mRNA 또는 단백질 발현 수준이 대조군 세포에 비해 증가된 세포 시료에 치료제 후보물질을 처리하는 단계; 및(a) ADAMTSL2 (ADAMTS-like protein 2), Cp (Ceruloplasmin), CFH (complement factor H), DDI2 (Protein DDI1 homolog2), FCN2 (Ficolin 2), IGFBP2 (insulin like growth factor binding protein 2), LGALS3BP ( galectin-3-binding protein), MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), THBS1 (Thrombospondin-1) And ZG16B (Zymogen granule protein 16 homolog B) comprising: treating a candidate therapeutic agent in a cell sample in which the mRNA or protein expression level for at least one marker selected from the group consisting of the control cells is increased compared to the control cells; And(b) 상기 후보 물질 처리 후, 상기 마커의 mRNA 또는 단백질 발현 수준이 감소되었는지를 확인하는 단계를 포함하는 실명 유발 안질환 치료용 물질의 스크리닝 방법.(b) After the treatment of the candidate substance, a method for screening a substance for treating blindness-causing eye diseases, comprising the step of checking whether the mRNA or protein expression level of the marker is decreased.
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001006262A1 (en) * | 1999-02-19 | 2001-01-25 | University Of Iowa Research Foundation | Diagnostics and therapeutics for macular degeneration |
US20120282285A1 (en) * | 2009-07-17 | 2012-11-08 | Rigshospitalet | Masp isoforms as inhibitors of complement activation |
KR101343285B1 (en) * | 2012-01-03 | 2013-12-18 | 서울대학교산학협력단 | Markers for Early Diagnosing of Diabetic Retinopathy and Use Thereof |
KR20140101134A (en) * | 2013-02-08 | 2014-08-19 | 건국대학교 산학협력단 | Method for providing information by Proteomic Analysis of the Aqueous Humor in Age-related Macular Degeneration Patients and biomarker for Age-related Macular Degeneration |
EP2770326A1 (en) * | 2013-02-26 | 2014-08-27 | Instituto Oftalmologico Fernandez - Vega, S.L. | Method for the diagnosis of glaucoma based on the determination of serum protein levels |
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WO2001006262A1 (en) * | 1999-02-19 | 2001-01-25 | University Of Iowa Research Foundation | Diagnostics and therapeutics for macular degeneration |
US20120282285A1 (en) * | 2009-07-17 | 2012-11-08 | Rigshospitalet | Masp isoforms as inhibitors of complement activation |
KR101343285B1 (en) * | 2012-01-03 | 2013-12-18 | 서울대학교산학협력단 | Markers for Early Diagnosing of Diabetic Retinopathy and Use Thereof |
KR20140101134A (en) * | 2013-02-08 | 2014-08-19 | 건국대학교 산학협력단 | Method for providing information by Proteomic Analysis of the Aqueous Humor in Age-related Macular Degeneration Patients and biomarker for Age-related Macular Degeneration |
EP2770326A1 (en) * | 2013-02-26 | 2014-08-27 | Instituto Oftalmologico Fernandez - Vega, S.L. | Method for the diagnosis of glaucoma based on the determination of serum protein levels |
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