Disease-specific biomarker for predicting and diagnosing pre-eclampsia in mid-pregnancy and use thereof

Abstract

The present invention relates to a disease-specific biomarker for early prediction and diagnosis of pre-eclampsia and a use thereof. A composition for predicting or diagnosing pre-eclampsia or a method for providing information for such prediction or diagnosis according to an aspect can simply and effectively predict or diagnose a disease by measuring and comparing the level of a disease-specific protein that changes in patients or the mRNA level of a gene encoding the protein.

Description

Disease-specific biomarkers for predicting and diagnosing preeclampsia in the second trimester of pregnancy and their uses

[0001] This relates to disease-specific biomarkers and their uses for early prediction and diagnosis of preeclampsia.

[0002] Pre-eclampsia (PE) is a condition characterized by hypertension (>140 / 90 mmHg) and proteinuria (>300 mg / day) occurring after 20 weeks of pregnancy, caused by various factors such as inflammation, hypoxia, and oxidative stress. It is a significant cause of maternal mortality, occurring in 7-12% of pregnant women, and is also responsible for a significant proportion of fetal and neonatal deaths and morbidities. Severe cases can lead to maternal pulmonary edema, cerebral hemorrhage, liver and kidney failure, blood clotting abnormalities, and even death. Furthermore, it can impair placental and fetal blood flow, leading to fetal growth retardation and, in severe cases, fetal death. However, the exact cause and treatment of PE remain unclear, and the only treatment option is delivery, which allows the mother to escape the pregnancy. However, because pre-eclampsia can increase the risk of premature birth and fetal mortality, early diagnosis and prolonged delivery are the optimal treatment.

[0003] In pregnant women after 20 weeks of pregnancy (multiple pregnancies after 32 weeks), diagnosis is possible using various indicators such as hypertension, proteinuria, and edema, but an early diagnosis method for pre-eclampsia to reduce premature birth caused by pre-eclampsia has not yet been clearly identified. Currently, in the case of a diagnosis method for pre-eclampsia, a test using a plasma marker has been developed, but the PerkinElmer DELFIA® Xpress PlGF kit has a false positive rate of 5% and a detection rate of 44%, and the Alere Triage® PlGF test has insufficient clinical diagnostic utility, so it still has low accuracy and insufficient clinical utility.

[0004] Accordingly, in order to prevent preeclampsia and improve the prognosis through treatment during the disease, a testing method that can effectively predict preeclampsia in the early and mid-terms of pregnancy and simplify diagnosis in the mid and late-terms of pregnancy is needed, but no conventional technology has been developed to date.

[0005] Accordingly, the inventors of the present invention have discovered the world's first preeclampsia-specific biomarker that shows significant changes in patients, and have developed a simple and effective method for early prediction and diagnosis of preeclampsia.

[0006] One aspect is to provide a biomarker composition for predicting or diagnosing preeclampsia in early, mid or late pregnancy, comprising a protein or a gene encoding the same.

[0007] Another aspect provides for the use of a biomarker comprising a protein or a gene encoding the same for the prediction or diagnosis of pre-eclampsia in early, mid or late pregnancy.

[0008] Another aspect provides a composition for predicting or diagnosing preeclampsia in early, middle or late pregnancy, comprising a preparation for measuring the level of mRNA of the protein or the gene encoding it.

[0009] Another aspect provides a use of a preparation for measuring the level of mRNA of the protein or the gene encoding it for the prediction or diagnosis of pre-eclampsia in early, middle or late pregnancy.

[0010] Another aspect provides a use for preparing a composition for predicting or diagnosing preeclampsia in early, middle or late pregnancy, comprising a preparation measuring the level of mRNA of the protein or the gene encoding it.

[0011] Another aspect is to provide a kit for predicting or diagnosing preeclampsia in early, middle or late pregnancy, comprising the composition for predicting or diagnosing preeclampsia.

[0012] Another aspect provides a method for providing information for predicting or diagnosing pre-eclampsia in early, mid or late pregnancy, comprising the steps of: measuring the level of mRNA of the protein or the gene encoding it in a sample isolated from a subject in early, mid or late pregnancy; and comparing the level of mRNA of the protein or the gene encoding it with a normal control sample in early, mid or late pregnancy.

[0013] Another aspect provides a method for predicting or diagnosing pre-eclampsia in early, mid or late pregnancy, comprising the steps of: measuring the level of mRNA of the protein or the gene encoding it in a sample isolated from a subject in early, mid or late pregnancy; and comparing the level of mRNA of the protein or the gene encoding it with a normal control sample in early, mid or late pregnancy.

[0014] One aspect provides a biomarker composition for predicting or diagnosing preeclampsia in early, mid or late pregnancy, comprising a protein or a gene encoding the same.

[0015] Another aspect provides for the use of biomarkers comprising proteins or genes encoding same for the prediction or diagnosis of pre-eclampsia in early, mid or late pregnancy.

[0016] Another aspect provides a composition for predicting or diagnosing preeclampsia in early, middle or late pregnancy, comprising a preparation for measuring the level of mRNA of the protein or the gene encoding it.

[0017] Another aspect provides the use of a formulation for measuring the level of mRNA of the protein or the gene encoding it for the prediction or diagnosis of pre-eclampsia in early, middle or late pregnancy.

[0018] Another aspect provides a use for preparing a composition for predicting or diagnosing preeclampsia in early, middle or late pregnancy, comprising a preparation measuring the level of mRNA of the protein or the gene encoding it.

[0019] In a biomarker composition for predicting or diagnosing pre-eclampsia in early pregnancy according to one specific example or a composition for predicting or diagnosing pre-eclampsia in early pregnancy, the protein may include one or more proteins selected from the group consisting of APOE, MMP2, LTA4H, ENG, GPT, SERPINB3, KRT9, CHI3L1, DSC2, SPP2, GFUS, CD109, SCARA5, MYOC, ADA2, PSMD9, TREH, SERPINA7, TUBB, PFN1, HLA-C, G6PD, ANXA3, RAN, CTBS, GDF15, XYLT2 and GP6.

[0020] In a biomarker composition for predicting or diagnosing mid-trimester pre-eclampsia according to one specific example or a composition for predicting or diagnosing mid-trimester pre-eclampsia, the protein may include one or more proteins selected from the group consisting of IGLC2, IGLC3, IGLC6, IGLC7, PLXNA2, GBA, ALDOB, COL1A2, MMP9, EPHA1, PTPRB, GSTO1, AOC3, ERAP2, SEZ6L2, ADAMTSL4, SCARA5, VAT1, CPVL, PSG8, ARID1B, WIPF1, CP, AGRN, CES2, TREH, APOB, SERPINE2, ITGA5, F7, HLA-C, GHR, LIPC, CAST, PRCP, ADAM17, FLNC, NOTUM and VWA1.

[0021] In a biomarker composition for diagnosing late-stage pre-eclampsia or a composition for diagnosing late-stage pre-eclampsia according to one specific example, the protein is GSK3A, PSMA7, SUSD5, CFD, COL3A1, FN1, FTL, CAT, SOD2, ALDOB, APP, CA3, DCN, MMP2, ITGA5, PAEP, ESD, COL6A2, F5, CEACAM1, GOT1, FLT1, SDC1, CAST, COL5A1, PEBP1, SDC4, HSPA4, CHI3L1, APLP1, FBLN2, TRAP1, SELENBP1, GFUS, POSTN, CSPG4, ADAMTSL4, CEMIP, KCTD12, COLEC11, PAPPA2, SEZ6L, CDH23, GKN1, CRYBB3, ADAM12, TREH, It may include one or more proteins selected from the group consisting of SEMA7A, CLN5, AGT, C9, LRG1, TTR, HPX, CLEC3B, SERPINA7, SERPIND1, C2, GUSB, CSF1, C4A, C4B, HLA-C, PTPRF, PSG2, LBP, FBLN1, CDH5, ENPEP, SOS1, PCDH1, DDR1, MGAT2, GALNT1, PTK7, DSG2, ADGRG6, ITIH5, OIT3, NRCAM, SEMA6A, DPEP2, CDHR5, IL1RAP, FETUB, PSG8, ST3GAL6 and PLXND1.

[0022] The term "pre-eclampsia (PE)" as used herein refers to hypertensive disorders associated with pregnancy, specifically gestational hypertension, and may encompass more advanced forms such as pre-eclampsia and eclampsia. Symptoms of PE include headache, edema, nausea, vomiting, and foamy urine.

[0023] In one specific example, the pathological condition of the preeclampsia may be at least one selected from the group consisting of chronic hypertension, gestational hypertension, preeclampsia, eclampsia, or complicated preeclampsia.

[0024] The first trimester of pregnancy refers to the period from the first day of the last menstrual period to the 13th week and 6th day, the second trimester refers to the period from the first day of the 14th week to the 27th week and 6th day, and the third trimester refers to the period from the first day of the 28th week to the 42nd week.

[0025] The term “diagnosis” herein may refer to determining a subject’s susceptibility to a particular disease or condition, determining whether a subject has a particular disease or condition, determining a prognosis for a subject with a particular disease or condition, or monitoring the same.

[0026] Previously, preeclampsia could only be diagnosed after the 20th week of pregnancy, but according to the composition for predicting or diagnosing preeclampsia or the method for providing information for predicting or diagnosing the same according to the present invention, preeclampsia can be effectively predicted in the early and mid-terms of pregnancy and diagnosis in the mid and late-terms of pregnancy can be simplified.

[0027] In this specification, the term “marker” or “biomarker” means a substance that can be evaluated to distinguish between individuals with a disease and individuals without the disease, and may include organic biomolecules such as polypeptides, proteins, nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), metabolites, etc., which show an increase or decrease in individuals with the disease compared to individuals without the disease.

[0028] The term “level measurement” herein may include measuring the extent of expression or activity of a marker for preeclampsia in a biological sample to predict or diagnose preeclampsia.

[0029] Specifically, measuring the level of the protein may mean measuring the degree of expression of the protein that changes with the onset of preeclampsia in a pregnant woman in the early, middle or late trimester of pregnancy.

[0030] Specifically, measuring the level of the mRNA may mean measuring the degree of expression of the mRNA that changes according to the onset of preeclampsia in a pregnant woman in the early, middle or late trimester of pregnancy.

[0031]

[0032] In one embodiment, the agent for measuring the level of the protein may comprise one or more selected from the group consisting of antibodies, interacting proteins, oligopeptides, ligands, nanoparticles, and aptamers that specifically bind to the protein or a peptide fragment thereof.

[0033] The above protein may include not only the protein itself, but also protein isoforms or protein variants that may be produced by splicing and variable promoters, or may be produced due to genetic changes such as mutations or polymorphisms.

[0034] The antibody may refer to a substance that specifically binds to the protein and causes an antigen-antibody reaction. The antibody may include a polyclonal antibody, a monoclonal antibody, or a recombinant antibody. The antibody may be readily produced using techniques well known in the art. The antibody may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. In addition, the antibody may include a complete form having two full-length light chains and two full-length heavy chains, as well as a functional fragment of the antibody molecule.

[0035] The above-mentioned interacting protein refers to a protein having a protein-protein interaction (PPI) with the above-mentioned protein, and may refer to a protein having a highly specific physical contact as a result of a biochemical action regulated by interactions including electrostatic force, hydrogen bonding, and hydrophobic effect.

[0036] The above oligopeptides are peptides composed of 2 to 20 amino acids and may include, but are not limited to, dipeptides, tripeptides, tetrapeptides and pentapeptides.

[0037] The above aptamer refers to a single-stranded oligonucleotide, and may refer to a nucleic acid molecule having binding activity to the protein. The aptamer may have various secondary or tertiary structures depending on its base sequence, and may have high affinity for a specific substance, such as an antigen-antibody reaction. The aptamer may be RNA, DNA, a modified nucleic acid, or a mixture thereof, and may be linear or cyclic in shape.

[0038]

[0039] In one specific example, the agent for measuring the level of mRNA of the gene may include one or more selected from the group consisting of a primer, a probe, a peptide nucleic acid (PNA), and an antisense nucleotide that specifically binds to the gene.

[0040] The above primer is a fragment that recognizes a target gene sequence, and may include a pair of forward and reverse primers. Preferably, the primer may be a pair of primers that provide analysis results with specificity and sensitivity. When the nucleic acid sequence of the primer is a sequence that does not match the non-target sequence present in the sample, and thus the primer only amplifies the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification, high specificity can be imparted.

[0041] The above probe refers to a nucleic acid fragment such as RNA or DNA that can specifically bind to a target nucleic acid, for example, mRNA, and may be labeled so as to confirm the presence or absence, content, and expression level of a specific mRNA. The probe may be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. The selection of an appropriate probe and hybridization conditions may be appropriately selected according to techniques known in the art.

[0042] The above PNA refers to an artificially synthesized polymer similar to DNA or RNA. While DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which can greatly increase binding affinity and stability to DNA or RNA of a target sequence.

[0043] The above antisense may refer to an oligomer having a sequence of nucleotide bases and an intersubunit backbone that allows the antisense oligomer to hybridize with a target sequence within RNA by Watson-Crick base pairing, typically forming an mRNA and RNA:oligomer heteroduplex within the target sequence. The oligomer may have exact sequence complementarity or approximate sequence complementarity to the target sequence.

[0044] Since the mRNA information of the protein or the gene encoding it according to the present invention is known in the art, a person skilled in the art can design a preparation for measuring the level of mRNA of the protein or the gene encoding it based on this.

[0045]

[0046] Another aspect provides a kit for predicting or diagnosing preeclampsia in early, middle or late pregnancy, comprising the composition for predicting or diagnosing preeclampsia.

[0047] In one specific example, the kit may be, but is not limited to, an ELISA kit, a protein chip kit, a rapid kit, a multiple reaction monitoring (MRM) kit, an RT-PCR kit, or a DNA chip kit.

[0048] The kit may further comprise one or more other component compositions, solutions or devices suitable for the analytical method.

[0049] The ELISA kit may include the essential components necessary for performing an ELISA. The ELISA kit may include an antibody specific for the protein. The antibody is an antibody with high specificity and affinity for the marker protein and little cross-reactivity with other proteins, and may be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. The ELISA kit may also include an antibody specific for a control protein (e.g., a housekeeping protein). In addition, the ELISA kit may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies), and their substrates or other substances capable of binding to antibodies.

[0050] The above protein chip kit may include essential components necessary for performing a protein chip. The protein chip kit may include a substrate to which a protein or fragment thereof is attached, and reagents, preparations, enzymes, etc. for producing a fluorescently labeled probe. The substrate may also include, but is not limited to, a control protein or fragment thereof.

[0051] The above rapid kit may be composed of, but is not limited to, a sample pad to which a sample is applied, a membrane to which an antibody is immobilized, and an absorption pad capable of absorbing the sample.

[0052] The above MRM kit may further include essential components required for performing multiple reaction monitoring. The MRM kit may include a peptide that selectively recognizes the protein.

[0053] The above RT-PCR kit may further include essential components required for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit may include a primer pair specific for the gene. The primers are nucleotides having a sequence specific to the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include a primer specific to the nucleic acid sequence of a control gene (e.g., a housekeeping gene). In addition, the reverse transcription polymerase reaction kit may include, but is not limited to, a test tube or other appropriate container, a reaction buffer (with various pH and magnesium concentrations), deoxynucleotides (dNTPs), an enzyme such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

[0054] The above DNA chip kit may include essential components necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or a fragment thereof are attached, and reagents, agents, enzymes, etc. for producing a fluorescently labeled probe. The substrate may also include, but is not limited to, cDNA or oligonucleotides corresponding to a control gene (e.g., a housekeeping gene) or a fragment thereof.

[0055] In addition, the kit may be an integrated magneto-electrochemical sensor kit. The integrated magneto-electrochemical sensor refers to a system that detects an electrical signal by enzymatic signal amplification using magnetic beads. Specifically, it may refer to a system that detects an electrical signal by enzymatic signal amplification generated by attaching a substance (e.g., an antibody) capable of detecting a target substance to a magnetic bead and a chromogenic substance capable of detecting the target substance (e.g., horseradish peroxidase (HRP), alkaline phosphatase (ALP), α-D-galactosidase (α-Gal)).

[0056] In one specific example, the kit may include a preparation, a device, and a computer having an algorithm built in for measuring the level of mRNA of the protein or a gene encoding the protein, and may relate to a kit that associates the result of measuring the level of the marker with the prediction or diagnosis of preeclampsia through the algorithm.

[0057]

[0058] Another aspect provides a method for providing information for predicting or diagnosing pre-eclampsia in early, mid, or late pregnancy, comprising the steps of: measuring the level of mRNA of the protein or the gene encoding it in a sample isolated from a subject in early, mid, or late pregnancy; and comparing the level of mRNA of the protein or the gene encoding it with a normal control sample in early, mid, or late pregnancy.

[0059] Another aspect provides a method for predicting or diagnosing pre-eclampsia in early, mid or late pregnancy, comprising the steps of measuring the level of mRNA of the protein or the gene encoding it in a sample isolated from a subject in early, mid or late pregnancy; and comparing the level of mRNA of the protein or the gene encoding it with a normal control sample in early, mid or late pregnancy.

[0060] The term “subject” in this specification refers to a mammal including a human, and may be selected from the group consisting of, for example, a human, a rat, a mouse, a guinea pig, a hamster, a rabbit, a monkey, a dog, a cat, a cow, a horse, a pig, a sheep, and a goat, and is preferably a human, but is not limited thereto.

[0061] The term “sample” as used herein means any material, biological fluid, tissue or cell obtained from or derived from a subject who is at risk for preeclampsia, has developed preeclampsia or is suspected of developing preeclampsia, for example, blood including whole blood, leukocytes, peripheral blood mononuclear cells, leukocyte buffy coat, plasma and serum, sputum, tears, mucus, nasal fluid, nasal aspirate, breath, urine, semen, saliva, peritoneal lavage, pelvic fluid, cyst fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, tracheal secretions, cells, cell extracts or cerebrospinal fluid, but preferably, it may be a liquid biopsy, for example, blood, serum or plasma.

[0062] According to one specific example, the method of the present invention may be performed on a sample separated from a subject by appropriately processing the sample (e.g., removing abundant protein, peptidization, etc.) according to a method known in the art to measure the level of a protein of a marker or mRNA of a gene encoding the protein, but is not limited thereto.

[0063] In a method for providing information for predicting or diagnosing pre-eclampsia in early pregnancy according to one specific example, the protein may include one or more proteins selected from the group consisting of APOE, MMP2, LTA4H, ENG, GPT, SERPINB3, KRT9, CHI3L1, DSC2, SPP2, GFUS, CD109, SCARA5, MYOC, ADA2, PSMD9, TREH, SERPINA7, TUBB, PFN1, HLA-C, G6PD, ANXA3, RAN, CTBS, GDF15, XYLT2 and GP6.

[0064] In a method for providing information for predicting or diagnosing pre-eclampsia in the second trimester of pregnancy according to one specific example, the protein may include at least one protein selected from the group consisting of IGLC2, IGLC3, IGLC6, IGLC7, PLXNA2, GBA, ALDOB, COL1A2, MMP9, EPHA1, PTPRB, GSTO1, AOC3, ERAP2, SEZ6L2, ADAMTSL4, SCARA5, VAT1, CPVL, PSG8, ARID1B, WIPF1, CP, AGRN, CES2, TREH, APOB, SERPINE2, ITGA5, F7, HLA-C, GHR, LIPC, CAST, PRCP, ADAM17, FLNC, NOTUM, and VWA1.

[0065] In a method for providing information for diagnosing pre-eclampsia in late pregnancy according to one specific example, the protein is GSK3A, PSMA7, SUSD5, CFD, COL3A1, FN1, FTL, CAT, SOD2, ALDOB, APP, CA3, DCN, MMP2, ITGA5, PAEP, ESD, COL6A2, F5, CEACAM1, GOT1, FLT1, SDC1, CAST, COL5A1, PEBP1, SDC4, HSPA4, CHI3L1, APLP1, FBLN2, TRAP1, SELENBP1, GFUS, POSTN, CSPG4, ADAMTSL4, CEMIP, KCTD12, COLEC11, PAPPA2, SEZ6L, CDH23, GKN1, CRYBB3, ADAM12, TREH, SEMA7A, CLN5, AGT, It may include one or more proteins selected from the group consisting of C9, LRG1, TTR, HPX, CLEC3B, SERPINA7, SERPIND1, C2, GUSB, CSF1, C4A, C4B, HLA-C, PTPRF, PSG2, LBP, FBLN1, CDH5, ENPEP, SOS1, PCDH1, DDR1, MGAT2, GALNT1, PTK7, DSG2, ADGRG6, ITIH5, OIT3, NRCAM, SEMA6A, DPEP2, CDHR5, IL1RAP, FETUB, PSG8, ST3GAL6 and PLXND1.

[0066] In one specific example, the step of measuring the level of the protein may be performed by at least one selected from the group consisting of protein mass spectrometry, protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, aukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry), Western blot, and ELISA (Enzyme Linked Immunosorbent Assay).

[0067] In one specific example, the step of measuring the level of mRNA of the gene may be performed by at least one selected from the group consisting of reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, and DNA chip.

[0068] In one specific example, the method for providing information for predicting or diagnosing preeclampsia in early pregnancy may further include a step of determining preeclampsia when the level of mRNA of one or more proteins selected from the group consisting of APOE, MMP2, LTA4H, ENG, GPT, SERPINB3, KRT9, CHI3L1, DSC2, SPP2, GFUS, CD109, SCARA5, MYOC, and ADA2 or a gene encoding the same is increased compared to a control group, or determining preeclampsia when the level of mRNA of one or more proteins selected from the group consisting of PSMD9, TREH, SERPINA7, TUBB, PFN1, HLA-C, G6PD, ANXA3, RAN, CTBS, GDF15, XYLT2, and GP6 or a gene encoding the same is decreased compared to a control group.

[0069] In one specific example, the method for providing information for predicting or diagnosing pre-eclampsia in the second trimester comprises determining pre-eclampsia when the level of mRNA of one or more proteins selected from the group consisting of IGLC2, IGLC3, IGLC6, IGLC7, PLXNA2, GBA, ALDOB, COL1A2, MMP9, EPHA1, PTPRB, GSTO1, AOC3, ERAP2, SEZ6L2, ADAMTSL4, SCARA5, VAT1, CPVL and PSG8 or a gene encoding the same is increased compared to the control group, or determining that the level of mRNA of one or more proteins selected from the group consisting of ARID1B, WIPF1, CP, AGRN, CES2, TREH, APOB, SERPINE2, ITGA5, F7, HLA-C, GHR, LIPC, CAST, PRCP, ADAM17, FLNC, NOTUM and VWA1 or a gene encoding the same is increased compared to the control group. If it is reduced, it may include an additional step to determine if it is preeclampsia.

[0070] In one specific example, the method for providing information for diagnosing pre-eclampsia in the late stage of pregnancy comprises: determining pre-eclampsia when the level of mRNA of one or more proteins selected from the group consisting of GSK3A, PSMA7, SUSD5, CFD, COL3A1, FN1, FTL, CAT, SOD2, ALDOB, APP, CA3, DCN, MMP2, ITGA5, PAEP, ESD, COL6A2, F5, CEACAM1, GOT1, FLT1, SDC1, CAST, COL5A1, PEBP1, SDC4, HSPA4, CHI3L1, APLP1, FBLN2, TRAP1, SELENBP1, GFUS, POSTN, CSPG4, ADAMTSL4, CEMIP, KCTD12, COLEC11, PAPPA2, SEZ6L, CDH23 and GKN1 or a gene encoding the same is increased compared to the control group; The method may further include a step of determining preeclampsia when the level of mRNA of one or more proteins selected from the group consisting of CRYBB3, ADAM12, TREH, SEMA7A, CLN5, AGT, C9, LRG1, TTR, HPX, CLEC3B, SERPINA7, SERPIND1, C2, GUSB, CSF1, C4A, C4B, HLA-C, PTPRF, PSG2, LBP, FBLN1, CDH5, ENPEP, SOS1, PCDH1, DDR1, MGAT2, GALNT1, PTK7, DSG2, ADGRG6, ITIH5, OIT3, NRCAM, SEMA6A, DPEP2, CDHR5, IL1RAP, FETUB, PSG8, ST3GAL6 and PLXND1 or a gene encoding the same is reduced compared to the control group.

[0071] As used herein, the term “increased protein level” or “increased mRNA level of a gene” means a measurable and significant increase in the level of protein or mRNA in a sample of a subject compared to a control, for example, an increase of about 1.1 times or more, for example, 1.1 to 2.5 times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, or 2 times or more.

[0072] The term “reduction in protein level” or “reduction in gene mRNA level” as used herein means a measurable and significant decrease in the level of protein or mRNA in a sample of a subject compared to a control, for example, a decrease of about 0.9 times or less, for example, 0.1 times to 0.9 times, 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, 0.4 times, 0.3 times, 0.2 times, or 0.1 times or less.

[0073] In one specific example, the method of providing information for predicting or diagnosing preeclampsia can be performed using a plurality of panels.

[0074] A composition for predicting or diagnosing preeclampsia according to a daily aspect or a method for providing information for predicting or diagnosing the same can simply and effectively predict or diagnose preeclampsia by measuring and comparing the level of a disease-specific protein that changes in a patient or the mRNA of a gene encoding the same.

[0075] Figure 1 shows the results of a heat map analysis of the levels of early pregnancy toxemia-specific biomarkers selected for provisional early pregnancy toxemia prediction or diagnosis.

[0076] Figure 2 shows the results of a heat map analysis of the levels of mid-trimester preeclampsia-specific biomarkers selected for the prediction or diagnosis of mid-trimester preeclampsia.

[0077] Figure 3 shows the results of a heat map analysis of the levels of late-stage preeclampsia-specific biomarkers selected for the diagnosis of late-stage preeclampsia.

[0078] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0079] Terms or words used in the specification and claims of the present invention are not to be construed as limited to their usual or dictionary meanings, and should be interpreted as meanings and concepts that conform to the technical idea of ​​the present invention based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0080] Throughout the specification of the present invention, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0081] Throughout the specification of the present invention, “A and / or B” means A or B, or A and B.

[0082]

[0083] Example 1. Extraction of plasma proteins from early, mid, and late pregnancy maternal blood.

[0084] Plasma proteins were extracted from maternal plasma samples from 10 normal pregnant women and 10 pregnant women with preeclampsia during early, mid, and late pregnancy.

[0085]

[0086] 1.1 Plasma depletion

[0087] Each plasma sample was diluted 4-fold with buffer A, centrifuged at 16,000 g for 1 minute using a 0.22 ㎛ spin filter, and depleted using a Nexera HPLC system and a multi affinity removal column (5188-6558) under the HPLC conditions in Table 1 below to extract protein samples. The protein samples were then recovered using a fraction collector and vacuum-dried using a SpeedVac.

[0088]

[0089] TimeModuleCommandValue0.01PumpsFlow rate0.1250.33ValveClose-8.00ValveOplen-17.00ValveClose-18.00PumpsFlow rate0.12518.01PumpsFlow rate120.00Pumps% B buffer020.01Pumps% B buffer10027.00Pumps% B buffer10027.01Pumps% B buffer038.00ControlerStop-

[0090]

[0091] 1.2 Protein lysis and digestion

[0092] Protein dissolution and digestion are performed in the following steps (1) to (4).

[0093] (1) Protein lysis: 100 μl of lysis buffer (1% SDS in 50 mM TEAB) was added to the dried sample and sonicated for 4 minutes. The sonicated sample was vortexed and spun down, and then sonicated again for 4 minutes.

[0094] (2) Protein reduction and alkylation: A final concentration of 10 mM DTT (dithiothreitol) was added to each sample, placed in a thermomixer, and a reduction reaction was performed at 56 °C for 30 minutes. After the reaction was completed, a final concentration of 40 mM IAA (iodoacetamide) was added, and an alkylation reaction was performed at room temperature in a dark room for 30 minutes.

[0095] (3) S-TRAP Protein Digestion: A final concentration of 1.2% phosphoric acid solution was added to the alkylated sample, and binding buffer (100 mM TEAB in 90% methanol) corresponding to 7 times the lysis buffer was added. The sample was loaded onto an S-trap column and centrifuged at 1,000 g for 1 minute. The S-trap column was washed three times with 400 μl of binding buffer, and 0.4 μg / μl of Promega trypsin (sequencing grade) was added to 125 μl of digestion buffer (50 mM TEAB) at a 1:25 = enzyme:protein ratio, and the reaction was performed in a 37 °C incubator for 16 hours.

[0096] (4) Elusion & Reconstitution: 80 μl of 50 mM TEAB was added to the S-trap column where enzyme digestion was completed, and the peptide sample was centrifuged at 1,000 g for 1 minute. The sample was transferred to a new tube. 80 μl of 0.2% formic acid in water was added to the S-trap column, and the peptide sample was centrifuged at 1,000 g for 1 minute. The peptide sample was collected. 80 μl of 0.2% formic acid in 50% ACN was added to the S-trap column, and the peptide sample was centrifuged at 1,000 g for 1 minute. The collected peptide sample was vacuum-dried using a SpeedVac. The dried sample was reconstituted in a 0.1% TFA solution, and the peptide concentration was measured using a Nanodrop.

[0097]

[0098] Example 2. Selection of biomarkers for predicting or diagnosing preeclampsia in early pregnancy.

[0099] To select a biomarker for predicting or diagnosing preeclampsia in early pregnancy, analysis was performed using mass spectrometry-based LC / MS after protein extraction from maternal plasma in early pregnancy according to Example 1.

[0100] Specifically, mass spectrometric analysis was performed using a Nano LC-MS / MS (Thermo Dionex Ultimate 3000 coupled with Thermo Orbitrap Exploris 480) system according to the methods and conditions in Table 2 below.

[0101]

[0102] LC & LC-MS / MS systemNano-LC Ultimate 3000 & Thermo Orbitrap Exploris 480ColumnTrap column: Acclaim PepMap TM 100, 100 ㎛ x 2 ㎝Analytical column: PepMap TM RSLC C18, 75 ㎛ x 15 ㎝Column Temp.50 ℃Flow rate1 ㎕ / minInjection volume2 ㎍Mobile phaseA: 0.1 % formic acid in water + 5 % DMSO,B: 0.1 % formic acid in 80 % acetonitrile + 5 % DMSOGradientTime (min)Solvent A (%)Solvent B (%)09555851540703046604047595515955295560955IonizationESI positive-ion modeSpray voltage2.5 kVIon transfer tube temp.275 °CMS1 resolution60,000Scan range350-1,500 m / zFAIMS VoltagesOnFAIMS Compensation voltage-35 CVMS1 AGC target500 %MS max IT20 msMS2 resolution15,000Precursor mass range385-1,015 m / zIsolation window9 m / zWindow overlap0 m / zFAIMS Compensation voltage-35 CV-45 CVLoop control20 spectra20 spectraMS2 AGC target300 %MS max IT25 msMS2 NCE28 %

[0103]

[0104] For LC-MS / MS data analysis, data independent analysis (DIA) was performed on the results, and a database search was performed using DIA-NN, a software dedicated to DIA data analysis.

[0105]

[0106] Search parameters are as follows:

[0107] - Library-free mode (peptide length 7-50 from the UniProt human protein database, 1 or fewer missed cleavages, cysteine ​​alkylation and methionine oxidation set as fixed and variable modifications, respectively)

[0108] - MBR (match-between-runs) mode.

[0109]

[0110] Search results were filtered by precursor and protein-level FDR of 1%. Precursor-to-peptide-to-protein level summaries were median-valued, and quantile normalization at each level was used to fit the global distribution of all samples. To identify blood proteins (i.e., signatures) that were specifically up-regulated in each condition (normal and preeclampsia), protein signatures satisfying the following criteria (1) to (5) were defined:

[0111] When placed into two groups for comparison,

[0112] (1) In each group, only proteins with missing value <= 50% are retained.

[0113] (2) log2-fold-change is within the top 15% of the empirical null fold-change distribution created when the sample label is randomly shuffled 1,000 times.

[0114] (3) empirical p-value <0.05,

[0115] (4) median expression in the condition of interest >0, and

[0116] (5) median expression in the other conditions <0.

[0117]

[0118] As a result of the performance, the proteins showing final expression changes were selected as biomarkers for predicting or diagnosing preeclampsia in early pregnancy.

[0119]

[0120] Figure 1 shows the results of a heat map analysis of the levels of early pregnancy toxemia-specific biomarkers selected for provisional early pregnancy toxemia prediction or diagnosis.

[0121] As shown in Figure 1, APOE, MMP2, LTA4H, ENG, GPT, SERPINB3, KRT9, CHI3L1, DSC2, SPP2, GFUS, CD109, SCARA5, MYOC, and ADA2 showed relatively higher levels in the early pregnancy toxemia group compared to the control group.

[0122] For PSMD9, TREH, SERPINA7, TUBB, PFN1, HLA-C, G6PD, ANXA3, RAN, CTBS, GDF15, XYLT2, and GP6, relatively lower levels were observed in the early pregnancy toxemia group compared to the control group.

[0123]

[0124] Example 3. Selection of biomarkers for predicting or diagnosing mid-trimester preeclampsia.

[0125] To select a biomarker for predicting or diagnosing mid-trimester preeclampsia, analysis was performed using mass spectrometry-based LC / MS after protein extraction from mid-trimester maternal plasma according to Example 1.

[0126] The protein analysis was performed in the same manner as in Example 2 above. As a result of the performance, the protein showing the final expression change was selected as a biomarker for predicting or diagnosing mid-trimester preeclampsia.

[0127]

[0128] Figure 2 shows the results of a heat map analysis of the levels of mid-trimester preeclampsia-specific biomarkers selected for the prediction or diagnosis of mid-trimester preeclampsia.

[0129] As shown in Figure 2, IGLC2, IGLC3, IGLC6, IGLC7, PLXNA2, GBA, ALDOB, COL1A2, MMP9, EPHA1, PTPRB, GSTO1, AOC3, ERAP2, SEZ6L2, ADAMTSL4, SCARA5, VAT1, CPVL and PSG8 showed relatively higher levels in the mid-pregnancy toxemia group compared to the control group.

[0130] For ARID1B, WIPF1, CP, AGRN, CES2, TREH, APOB, SERPINE2, ITGA5, F7, HLA-C, GHR, LIPC, CAST, PRCP, ADAM17, FLNC, NOTUM, and VWA1, relatively lower levels were observed in the mid-trimester preeclampsia group compared to the control group.

[0131]

[0132] Example 4. Selection of biomarkers for diagnosing preeclampsia in late pregnancy.

[0133] To select a biomarker for diagnosing preeclampsia in late pregnancy, protein extraction from maternal plasma in late pregnancy according to Example 1 was performed and then analyzed using mass spectrometry-based LC / MS.

[0134] The protein analysis was performed in the same manner as in Example 2 above. As a result of the performance, the protein showing the final expression change was selected as a biomarker for diagnosing preeclampsia in the late stage of pregnancy.

[0135]

[0136] Figure 3 shows the results of a heat map analysis of the levels of late-stage preeclampsia-specific biomarkers selected for the diagnosis of late-stage preeclampsia.

[0137] As shown in Figure 3, GSK3A, PSMA7, SUSD5, CFD, COL3A1, FN1, FTL, CAT, SOD2, ALDOB, APP, CA3, DCN, MMP2, ITGA5, PAEP, ESD, COL6A2, F5, CEACAM1, GOT1, FLT1, SDC1, CAST, COL5A1, PEBP1, SDC4, HSPA4, CHI3L1, APLP1, FBLN2, TRAP1, SELENBP1, GFUS, POSTN, CSPG4, ADAMTSL4, CEMIP, KCTD12, COLEC11, PAPPA2, SEZ6L, CDH23 and GKN1 showed relatively higher levels in the late-stage preeclampsia group compared to the control group.

[0138] For CRYBB3, ADAM12, TREH, SEMA7A, CLN5, AGT, C9, LRG1, TTR, HPX, CLEC3B, SERPINA7, SERPIND1, C2, GUSB, CSF1, C4A, C4B, HLA-C, PTPRF, PSG2, LBP, FBLN1, CDH5, ENPEP, SOS1, PCDH1, DDR1, MGAT2, GALNT1, PTK7, DSG2, ADGRG6, ITIH5, OIT3, NRCAM, SEMA6A, DPEP2, CDHR5, IL1RAP, FETUB, PSG8, ST3GAL6, and PLXND1, levels were relatively lower in the late-term pre-eclampsia group compared to the control group.

[0139]

[0140] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the experimental examples and embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

[0141]

[0142] The present invention relates to a disease-specific biomarker for early prediction and diagnosis of preeclampsia and its use, which can be used for early prediction and diagnosis of preeclampsia.

[0143] Related national research and development projects

[0144] - Assignment ID: 1465039723

[0145] - Assignment Number: RS-2022-KH129953

[0146] - Ministry name: Ministry of Health and Welfare

[0147] - Project Management (Professional) Organization Name: Korea Health Industry Development Institute

[0148] - Research Project Name: Research-Oriented Hospital Development

[0149] Research Project Name: Establishing a Platform to Address Unmet Pregnancy and Childbirth Medical Needs Based on Future Innovation Technology

[0150] - Name of the project performing organization: Bundang Cha Hospital

[0151] - Research period for the current year: January 1, 2023 - December 31, 2023

Claims

1. A biomarker composition for predicting or diagnosing mid-trimester pre-eclampsia, comprising at least one protein selected from the group consisting of IGLC2, IGLC3, IGLC6, IGLC7, PLXNA2, GBA, ALDOB, COL1A2, MMP9, EPHA1, PTPRB, GSTO1, AOC3, ERAP2, SEZ6L2, ADAMTSL4, SCARA5, VAT1, CPVL, PSG8, ARID1B, WIPF1, CP, AGRN, CES2, TREH, APOB, SERPINE2, ITGA5, F7, HLA-C, GHR, LIPC, CAST, PRCP, ADAM17, FLNC, NOTUM and VWA1 or a gene encoding the same.

2. A composition for predicting or diagnosing mid-trimester pre-eclampsia, comprising an agent for measuring the level of one or more proteins selected from the group consisting of IGLC2, IGLC3, IGLC6, IGLC7, PLXNA2, GBA, ALDOB, COL1A2, MMP9, EPHA1, PTPRB, GSTO1, AOC3, ERAP2, SEZ6L2, ADAMTSL4, SCARA5, VAT1, CPVL, PSG8, ARID1B, WIPF1, CP, AGRN, CES2, TREH, APOB, SERPINE2, ITGA5, F7, HLA-C, GHR, LIPC, CAST, PRCP, ADAM17, FLNC, NOTUM and VWA1 or an mRNA of a gene encoding the same.

3. A composition for predicting or diagnosing mid-trimester pre-eclampsia, wherein the agent for measuring the level of the protein according to claim 2 comprises at least one selected from the group consisting of antibodies, interacting proteins, oligopeptides, ligands, nanoparticles, and aptamers that specifically bind to the protein or a peptide fragment thereof.

4. A composition for predicting or diagnosing mid-trimester pre-eclampsia, wherein the agent for measuring the level of mRNA of the gene according to claim 2 comprises at least one selected from the group consisting of a primer, a probe, a peptide nucleic acid (PNA), and an antisense nucleotide that specifically bind to the gene.

5. A kit for predicting or diagnosing mid-trimester preeclampsia, comprising a composition according to any one of claims 2 to 4.

6. A kit for predicting or diagnosing mid-trimester pre-eclampsia according to claim 5, wherein the kit is an ELISA kit, a protein chip kit, a rapid kit, an MRM (multiple reaction monitoring) kit, an RT-PCR kit, or a DNA chip kit.

7. A step of measuring the level of mRNA of one or more proteins selected from the group consisting of IGLC2, IGLC3, IGLC6, IGLC7, PLXNA2, GBA, ALDOB, COL1A2, MMP9, EPHA1, PTPRB, GSTO1, AOC3, ERAP2, SEZ6L2, ADAMTSL4, SCARA5, VAT1, CPVL, PSG8, ARID1B, WIPF1, CP, AGRN, CES2, TREH, APOB, SERPINE2, ITGA5, F7, HLA-C, GHR, LIPC, CAST, PRCP, ADAM17, FLNC, NOTUM and VWA1 in a sample isolated from a subject in the second trimester of pregnancy; and Comprising a step of comparing the level of mRNA of the protein or the gene encoding it with a normal control sample of the second trimester of pregnancy. A method for providing information for predicting or diagnosing pre-eclampsia during the second trimester of pregnancy.

8. In claim 7, the step of measuring the level of the protein comprises: A method for providing information for predicting or diagnosing mid-trimester pre-eclampsia, which is performed by at least one selected from the group consisting of protein mass spectrometry, protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, aukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry), western blot, and ELISA (Enzyme Linked Immunosorbentassay).

9. In claim 7, the step of measuring the level of mRNA of the gene comprises: A method for providing information for predicting or diagnosing pre-eclampsia of the second trimester, wherein the method is performed by at least one selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time RT-PCR, RNase protection assay (RPA), Northern blotting, and DNA chip.

10. In claim 7, if the level of mRNA of one or more proteins selected from the group consisting of IGLC2, IGLC3, IGLC6, IGLC7, PLXNA2, GBA, ALDOB, COL1A2, MMP9, EPHA1, PTPRB, GSTO1, AOC3, ERAP2, SEZ6L2, ADAMTSL4, SCARA5, VAT1, CPVL and PSG8 or a gene encoding the same is increased compared to the control group, it is judged to be preeclampsia, or A method of determining preeclampsia further comprises a step of determining that the level of mRNA of one or more proteins selected from the group consisting of ARID1B, WIPF1, CP, AGRN, CES2, TREH, APOB, SERPINE2, ITGA5, F7, HLA-C, GHR, LIPC, CAST, PRCP, ADAM17, FLNC, NOTUM and VWA1 or a gene encoding the same is decreased compared to the control group. A method for providing information for predicting or diagnosing pre-eclampsia during the second trimester of pregnancy.

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