Disease-specific biomarkers for prediction and diagnosis of early-onset preeclampsia and uses thereof

Abstract

The present invention relates to a disease-specific biomarker for early prediction and diagnosis of preeclampsia and use thereof, and a composition for prediction or diagnosis of preeclampsia according to an aspect or a method of providing information for prediction or diagnosis thereof, can simply and effectively predict or diagnose the disease by measuring and comparing the mRNA level of a disease-specific protein or a gene encoding the protein changed in a patient.

Description

Technical Field

[0001] This relates to a disease-specific biomarker for early prediction and diagnosis of preeclampsia and its applications. Background Technology

[0002] Preeclampsia (PE) is a hypertension (>140 / 90 mmHg) disorder characterized by proteinuria (>300 mg / day) after 20 weeks of gestation, caused by various factors such as inflammation, hypoxia, and oxidative stress. It occurs in 7-12% of pregnant women and is a significant cause of maternal mortality, also accounting for a large proportion of fetal or neonatal deaths and morbidity. In severe cases, it can lead to maternal pulmonary edema, cerebral hemorrhage, liver and kidney failure, coagulation abnormalities, and even death. Furthermore, it can cause impaired blood flow to the placenta and fetus, leading to fetal growth restriction and, in severe cases, fetal death. However, the exact causes and treatments for PE are not yet fully understood; the only treatment is delivery. However, because preeclampsia can increase the fetal mortality rate due to premature birth, early diagnosis and delaying delivery as much as possible are considered the best approach.

[0003] For pregnant women after 20 weeks of gestation (more common after 32 weeks), various indicators such as hypertension, proteinuria, and edema can be used for diagnosis. However, a definitive method for early diagnosis of preeclampsia to reduce premature birth caused by preeclampsia has not yet been established. Currently, regarding diagnostic methods for preeclampsia, although tests using plasma biomarkers have been developed, the PerkinElmer DELFIA® Xpress PlGF kit shows a 5% false positive rate and a 44% detection rate. The Abbott / Alere Triage® PlGF test still lacks sufficient evidence for clinical diagnostic utility. Therefore, the actual situation remains one of low accuracy and insufficient clinical utility evidence.

[0004] Therefore, in order to prevent preeclampsia and improve prognosis through treatment when it occurs, there is an urgent need for a detection method that can effectively predict preeclampsia in the early and middle stages of pregnancy and simplify diagnosis in the middle and late stages of pregnancy. However, there is currently no developed existing technology for this.

[0005] To this end, the inventors discovered the world's first preeclampsia-specific biomarker that showed significant changes in patients and derived a simple and effective method for early prediction and diagnosis of preeclampsia. Summary of the Invention

[0006] Technical issues One aspect is to provide a biomarker composition for the prediction or diagnosis of preeclampsia in early, mid, or late pregnancy, comprising a protein or a gene encoding the protein.

[0007] On the other hand, it is used to provide a biomarker containing a protein or a gene encoding that protein for the prediction or diagnosis of preeclampsia in early, mid, or late pregnancy.

[0008] Another aspect is to provide a composition for the prediction or diagnosis of preeclampsia in early, mid, or late pregnancy, comprising an agent for measuring the mRNA level of the protein or the gene encoding the protein.

[0009] Another aspect is the use of a preparation for predicting or diagnosing preeclampsia in early, mid, or late pregnancy by measuring the mRNA level of said protein or the gene encoding said protein.

[0010] Another aspect is the use of a preparation for determining the mRNA level of the protein or the gene encoding the protein in preparation of a composition for the prediction or diagnosis of preeclampsia in early, mid, or late pregnancy.

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

[0012] Another aspect is to provide a method for providing information for the prediction or diagnosis of preeclampsia in early, mid, or late pregnancy, comprising: measuring the mRNA level of the protein or the gene encoding the protein in a sample isolated from a subject in early, mid, or late pregnancy; and comparing the mRNA level of the protein or the gene encoding the protein with a normal control sample in early, mid, or late pregnancy.

[0013] Another aspect is to provide a method for predicting or diagnosing preeclampsia in early, mid, or late pregnancy, comprising: measuring the mRNA level of the protein or the gene encoding the protein in a sample isolated from a subject in early, mid, or late pregnancy; and comparing the mRNA level of the protein or the gene encoding the protein with a normal control group sample in early, mid, or late pregnancy.

[0014] Technical solution On the one hand, a biomarker composition for predicting or diagnosing preeclampsia in early, mid, or late pregnancy is provided, comprising a protein or a gene encoding the protein.

[0015] On the other hand, it provides a biomarker containing a protein or a gene encoding the protein for the prediction or diagnosis of preeclampsia in early, mid, or late pregnancy.

[0016] In another aspect, a composition is provided for the prediction or diagnosis of preeclampsia in early, mid, or late pregnancy, comprising an agent for determining the mRNA level of said protein or the gene encoding said protein.

[0017] In another aspect, there is the use of a preparation for determining the mRNA level of said protein or the gene encoding said protein for the prediction or diagnosis of preeclampsia in early, mid, or late pregnancy.

[0018] In another aspect, there is the use of a preparation for determining the mRNA level of said protein or the gene encoding said protein in the preparation of a composition for the prediction or diagnosis of preeclampsia in early, mid, or late pregnancy.

[0019] According to one specific example, in a biomarker composition for predicting or diagnosing preeclampsia in early pregnancy, or in a composition for predicting or diagnosing preeclampsia in early pregnancy, the protein may comprise 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] According to one specific example, in a biomarker composition for predicting or diagnosing preeclampsia in mid-pregnancy or in a composition for predicting or diagnosing preeclampsia in mid-pregnancy, the protein may comprise 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] According to one specific example, in a biomarker composition for the diagnosis of preeclampsia in late pregnancy or in a composition for the diagnosis of preeclampsia in late pregnancy, the protein may comprise a protein selected from 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. One or more proteins from the group consisting of PAPPA2, SEZ6L, CDH23, GKN1, 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.

[0022] In this manual, the term "pre-eclampsia (PE)" refers to a hypertensive disorder that occurs during pregnancy. Specifically, it can include gestational hypertension, preeclampsia (a more advanced form of preeclampsia), and eclampsia. Symptoms of preeclampsia include headache, edema, nausea, vomiting, and foamy urine.

[0023] According to a specific example, the condition of preeclampsia can be selected from one or more of the following groups: chronic hypertension, gestational hypertension, preeclampsia, eclampsia, or superimposed preeclampsia.

[0024] The term "early pregnancy" refers to the period from the start of the last menstrual period to 13 weeks and 6 days, "mid pregnancy" refers to the period from 14 weeks to 27 weeks and 6 days, and "late pregnancy" refers to the period from 28 weeks to 42 weeks.

[0025] In this specification, the term "diagnosis" may refer to determining a subject's susceptibility to a specific disease or condition, determining whether a subject has a specific disease or condition, determining the prognosis of a subject with a specific disease or condition, or monitoring such a subject.

[0026] Previously, preeclampsia could only be diagnosed after 20 weeks of gestation. However, the composition for predicting or diagnosing preeclampsia according to the present invention, or the method for providing information for its prediction or diagnosis, can effectively predict preeclampsia in the early and middle stages of pregnancy and simplify diagnosis in the middle and late stages of pregnancy.

[0027] In this specification, the term "marker" or "biomarker" refers to a substance that can distinguish and evaluate diseased individuals from healthy individuals. It may include organic biomolecules that are increased or decreased in diseased individuals compared to healthy individuals, such as polypeptides, proteins, nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), metabolites, etc.

[0028] In this specification, the term "level determination" may include measuring the degree of expression or activity of the biomarker in a biological sample for the purpose of predicting or diagnosing preeclampsia.

[0029] Specifically, the protein level determination may refer to determining the expression level of the protein in pregnant women during the early, middle, or late stages of pregnancy, as the onset of preeclampsia progresses.

[0030] Specifically, the mRNA level measurement may refer to measuring the expression level of the mRNA in pregnant women during the early, middle, or late stages of pregnancy, as the onset of preeclampsia progresses.

[0031] According to one specific example, the formulation for determining the protein level 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 its peptide fragments.

[0032] The protein may include not only the protein itself, but also protein isoforms or variants that can be produced by splicing and variable promoters, or by genetic changes such as mutations or polymorphisms.

[0033] The antibody can refer to a substance that specifically binds to the protein and induces an antigen-antibody reaction. The antibody may include polyclonal antibodies, monoclonal antibodies, or recombinant antibodies. The antibody can be readily prepared using techniques widely known in the art. The antibody can be separated and purified using methods such as gel electrophoresis, dialysis, salting out, ion exchange chromatography, and affinity chromatography. Furthermore, the antibody may include not only a complete morphology with two full-length light chains and two full-length heavy chains, but also functional fragments of the antibody molecule.

[0034] The term "interacting protein" refers to a protein that has a protein-protein interaction (PPI), which can mean a protein that has a highly specific physical contact as a result of biochemical processes regulated by interactions including electrostatic forces, hydrogen bonds, and hydrophobic effects.

[0035] The oligopeptide is a peptide composed of 2 to 20 amino acids, and may include dipeptides, tripeptides, tetrapeptides and pentapeptides, but is not limited thereto.

[0036] The aptamer refers to a single-stranded oligonucleotide, which can mean a nucleic acid molecule that has binding activity to the protein. The aptamer may have various secondary or tertiary structures depending on its base sequence, and may exhibit high affinity for specific substances, similar to an antigen-antibody reaction. The aptamer may be RNA, DNA, modified nucleic acids, or mixtures thereof, and its morphology may be linear or circular.

[0037] According to one specific example, the preparation for determining the mRNA level of the gene may contain one or more selected from the group consisting of primers, probes, PNA (peptide nucleic acid), and antisense nucleotides that specifically bind to the gene.

[0038] The primers, as fragments that recognize target gene sequences, may include forward and reverse primer pairs, and preferably primer pairs that provide analytical results with specificity and sensitivity. Higher specificity can be achieved when the nucleic acid sequence of the primers does not match the non-target sequence present in the sample, thereby amplifying only the target gene sequence containing the complementary primer binding site without inducing non-specific amplification.

[0039] The probe refers to a nucleic acid fragment, such as RNA or DNA, that can specifically bind to a target nucleic acid, such as mRNA, and can be labeled to confirm the presence, content, and expression level of a specific mRNA. Probes can be fabricated as oligonucleotide probes, single-strand DNA probes, double-strand DNA probes, RNA probes, etc. The selection of appropriate probes and hybridization conditions can be made according to techniques known in this field.

[0040] The PNA refers to a synthetically produced polymer similar to DNA or RNA. DNA has a phosphate-ribose backbone, while PNA has a repeating N-(2-aminoethyl)-glycine backbone linked by peptide bonds, thus significantly increasing the binding force and stability to target DNA or RNA sequences.

[0041] The term "antisense" refers to an oligomer with a nucleotide base sequence and an inter-subunit backbone. This antisense oligomer hybridizes with the target sequence within RNA via Watson-Crick base pairing, thereby allowing the typical formation of an mRNA-RNA:oligomeric heteroduplex within the target sequence. The oligomer can exhibit precise or near-complementarity with the target sequence.

[0042] The mRNA information of the protein or the gene encoding the protein according to the present invention is known in the art, and therefore those skilled in the art can design preparations to determine the mRNA level of the protein or the gene encoding the protein based on this.

[0043] In another aspect, a kit for predicting or diagnosing preeclampsia in early, mid, or late pregnancy is provided, comprising the composition for predicting or diagnosing preeclampsia.

[0044] According to one specific example, the kit may be an ELISA (enzyme-linked immunosorbent assay) kit, a protein chip kit, a rapid detection kit, an MRM (multiple reaction monitoring) kit, an RT-PCR (reverse transcription polymerase chain reaction) kit, or a DNA chip kit, but is not limited thereto.

[0045] The kit may also contain one or more other component compositions, solutions or devices suitable for the analytical method.

[0046] The ELISA kit may contain the necessary elements for performing an ELISA. The ELISA kit may contain a specific antibody against the protein in question. The antibody, as an antibody with high specificity and affinity for the marker protein and virtually no cross-reactivity with other proteins, is a monoclonal antibody, polyclonal antibody, or recombinant antibody. In addition, the ELISA kit may contain a specific antibody against a control group (e.g., housekeeping protein) protein. Furthermore, the ELISA kit may contain reagents capable of detecting antibodies that have bound to it, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies) and their substrates, or other substances capable of binding to antibodies.

[0047] The protein chip kit may contain the necessary elements for performing protein chip analysis. The protein chip kit may contain a substrate with proteins or fragments thereof attached, as well as reagents, formulations, enzymes, etc., for preparing fluorescently labeled probes. Furthermore, the substrate may contain control proteins or fragments thereof, but is not limited thereto.

[0048] The rapid test kit may consist of a sample pad for applying the sample, a membrane immobilized with antibodies, and an absorbent pad capable of absorbing the sample, but is not limited thereto.

[0049] The MRM kit may further include the necessary elements for performing multiple reaction monitoring. The MRM kit may contain peptides that selectively recognize the proteins.

[0050] The RT-PCR kit may also contain the necessary elements for performing reverse transcription polymerase chain reaction. The reverse transcription polymerase chain reaction kit may contain specific primer pairs targeting the gene. The primers, as nucleotides with specific sequences targeting the nucleic acid sequence of the gene, may be approximately 7 bp to 50 bp in length, more preferably approximately 10 bp to 30 bp. Additionally, it may contain specific primers targeting the nucleic acid sequence of a control gene (e.g., a housekeeping gene). Furthermore, the reverse transcription polymerase chain reaction kit may contain test tubes or other suitable containers, reaction buffers (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase (deoxyribonuclease), RNase (ribonuclease) inhibitors, DEPC water, sterile water, etc., but are not limited to these.

[0051] The DNA microarray kit may contain the necessary elements for performing DNA microarray analysis. The DNA microarray kit may contain a substrate with cDNA or oligonucleotides attached to a gene or fragment thereof, as well as reagents, formulations, enzymes, etc., for preparing fluorescently labeled probes. Furthermore, the substrate may contain cDNA or oligonucleotides corresponding to a control gene (e.g., a housekeeping gene) or fragment thereof, but is not limited thereto.

[0052] Furthermore, the kit can be an integrated magneto-electrochemical sensor kit. An integrated magneto-electrochemical sensor refers to a system that uses magnetic beads to detect electrical signals amplified by enzyme-catalyzed signals. Specifically, it can refer to a system in which a substance capable of detecting a target substance (e.g., an antibody) is attached to the magnetic beads, and the system detects the electrical signal amplified by the enzyme-catalyzed signal generated by a chromogenic substance capable of detecting the target substance (e.g., horseradish peroxidase (HRP), alkaline phosphatase (ALP), α-D-galactosidase (α-Gal)).

[0053] According to one specific example, the kit may include a formulation, apparatus, and computer for determining the mRNA level of the protein or the gene encoding the protein, and for correlating the determination of the biomarker level with the prediction or diagnosis of preeclampsia via the algorithm.

[0054] In another aspect, a method is provided for providing information for the prediction or diagnosis of preeclampsia in early, mid, or late pregnancy, comprising: measuring the mRNA level of the protein or the gene encoding the protein in a sample isolated from a subject in early, mid, or late pregnancy; and comparing the mRNA level of the protein or the gene encoding the protein with a normal control sample in early, mid, or late pregnancy.

[0055] In another aspect, a method for predicting or diagnosing preeclampsia in early, mid, or late pregnancy is provided, comprising: measuring the mRNA level of the protein or the gene encoding the protein in a sample isolated from a subject in early, mid, or late pregnancy; and comparing the mRNA level of the protein or the gene encoding the protein with a normal control sample in early, mid, or late pregnancy.

[0056] In this specification, the term "test subject" refers to mammals, including humans, and may be selected from the group consisting of humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep and goats, preferably humans, but not limited thereto.

[0057] In this specification, the term "sample" means any substance, biological fluid, tissue, or cell obtained from or derived from a subject who is at risk of preeclampsia, a patient with preeclampsia, or a subject suspected of having preeclampsia. For example, it may be one or more of the following: whole blood, white blood cells, peripheral blood mononuclear cells, white blood cell layer, blood containing plasma and serum, sputum, tears, mucus, nasal secretions, nasal aspirate, respiratory gases, urine, semen, saliva, peritoneal lavage fluid, pelvic fluid, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic juice, lymph, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, tracheal secretions, cells, cell extracts, or cerebrospinal fluid, but preferably a liquid biopsy, such as blood, serum, or plasma.

[0058] According to a specific example of the method of this application, in order to determine the mRNA level of a biomarker protein or the gene encoding the protein in a sample isolated from a test subject, the sample may be appropriately processed according to methods known in the art (e.g., removal of high-abundance proteins, peptidetization, etc.), but is not limited thereto.

[0059] According to one specific example, in a method of providing information for the prediction or diagnosis of preeclampsia in early pregnancy, the protein may comprise 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.

[0060] According to one specific example, in a method of providing information for the prediction or diagnosis of preeclampsia in the second trimester, the protein may comprise 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.

[0061] According to a specific example, in a method for providing information for the diagnosis of preeclampsia in late pregnancy, the protein may comprise a subset selected from 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, SE One or more proteins from the group consisting of Z6L, CDH23, GKN1, 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.

[0062] According to a specific example, the step of determining protein levels can be performed by selecting from protein mass spectrometry, protein chip analysis, immunoassay, ligand binding analysis, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry), SELDI-TOF (Surface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry), radioimmunoassay, radioimmunodiffusion, Ouchterlony 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 blotting, and ELISA (Enzyme Linked Immunosorbent Assay). The assay shall be performed using one or more of the following methods: Immunosorbent assay (ELISA).

[0063] According to one specific example, the step of determining the mRNA level of a gene can be performed by any one or more of the following methods: reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Real-time RT-PCR), ribonuclease protection assay (RPA), Northern blotting, and DNA microarray.

[0064] According to a specific example, the method for providing information for predicting or diagnosing preeclampsia in early pregnancy may further include the following steps: when the mRNA level of one or more proteins or genes encoding the proteins selected from the group consisting of APOE, MMP2, LTA4H, ENG, GPT, SERPINB3, KRT9, CHI3L1, DSC2, SPP2, GFUS, CD109, SCARA5, MYOC, and ADA2 is increased compared to the control group, preeclampsia is determined; or when the mRNA level of one or more proteins or genes encoding the proteins selected from the group consisting of PSMD9, TREH, SERPINA7, TUBB, PFN1, HLA-C, G6PD, ANXA3, RAN, CTBS, GDF15, XYLT2, and GP6 is decreased compared to the control group, preeclampsia is determined.

[0065] According to a specific example, the method for providing information for predicting or diagnosing preeclampsia in mid-pregnancy may further include the following steps: when the mRNA level of one or more proteins or genes encoding the 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 is increased compared to the control group, preeclampsia is determined; or when the mRNA level of one or more proteins or genes encoding the 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 is decreased compared to the control group, preeclampsia is determined.

[0066] According to a specific example, the method for providing information for the diagnosis of preeclampsia in late pregnancy may further include the following steps: when the mRNA of one or more proteins or genes encoding such 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 is... Preeclampsia is diagnosed when the level of one or more proteins or the mRNA of the gene encoding the selected proteins (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) is increased compared to the control group; or preeclampsia is diagnosed when the level of one or more proteins or the mRNA of the gene encoding the selected protein is decreased compared to the control group.

[0067] In this specification, the terms "increased protein levels" or "increased mRNA levels" mean that, compared with the control group, the levels of protein or mRNA in the test subject's sample are significantly increased to a measurable level, such as an increase of about 1.1 times or more, for example, an increase of 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 more.

[0068] In this specification, the terms "reduced protein level" or "reduced mRNA level" mean that, compared with the control group, the level of protein or mRNA in the sample of the test subject is significantly reduced to a measurable level, such as a reduction of about 0.9 times or less, for example, a reduction of 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 less.

[0069] In one specific example, the method for providing information for the prediction or diagnosis of preeclampsia can be performed in the form of a panel of multiple biomarkers.

[0070] Invention Effects According to a composition for the prediction or diagnosis of preeclampsia or a method for providing information for its prediction or diagnosis, preeclampsia can be predicted or diagnosed simply and effectively by measuring and comparing the mRNA levels of disease-specific proteins or genes encoding such proteins that change in patients. Attached Figure Description

[0071] Figure 1 The results are from a heat map analysis of the levels of preeclampsia-specific biomarkers selected for prediction or diagnosis of preeclampsia in early pregnancy.

[0072] Figure 2 The results are from a heat map analysis of the levels of preeclampsia-specific biomarkers selected for prediction or diagnosis of preeclampsia in the second trimester.

[0073] Figure 3 The results are from a heat map analysis of the levels of preeclampsia-specific biomarkers selected for diagnosis of preeclampsia in late pregnancy. Detailed Implementation

[0074] Preferred embodiments are presented below to aid in understanding the present invention. However, these embodiments are provided merely to facilitate understanding of the invention, and the scope of the invention is not limited to these embodiments. Various modifications can be made to the embodiments, and therefore the embodiments are not limited to those disclosed below, but can be implemented in various forms.

[0075] The terms or words used in this specification and claims should not be limited to their usual or dictionary meanings, but should be interpreted in accordance with the principle that the inventors may appropriately define terms and concepts in order to best illustrate their invention, and should be interpreted as meanings and concepts consistent with the technical ideas of this invention.

[0076] Throughout this specification, when a part is referred to as "including" a constituent element, it does not exclude other constituent elements unless otherwise stated, but rather means that other constituent elements may also be included.

[0077] Throughout this invention, “A and / or B” means A or B, or A and B.

[0078] Example 1. Extraction of maternal plasma proteins in early, mid, and late pregnancy Plasma proteins were extracted from maternal plasma samples from 10 normal pregnant women and 10 women with preeclampsia during the first, second, and third trimesters of pregnancy.

[0079] 1.1 Plasma depletion Each plasma sample was diluted 4-fold with buffer A, centrifuged at 16,000g for 1 minute using a 0.22μm centrifuge filter, and then subjected to protein removal using a Nexera HPLC system and a multi affinity removal column (5188-6558) according to the HPLC conditions in Table 1 below. The protein samples were then recovered using a fraction collector and vacuum-dried by vacuum centrifugation.

[0080] Table 1

[0081] 1.2 Protein lysis and digestion Protein cleavage and digestion include the following steps (1) to (4).

[0082] (1) Protein lysis: Add 100 μL of lysis buffer (50 mM triethylammonium bicarbonate (TEAB) solution containing 1% sodium dodecyl sulfate (SDS)) to the dried sample and sonicate for 4 minutes; after sonication, vortex and centrifuge the sample and sonicate for another 4 minutes.

[0083] (2) Protein reduction and alkylation: Add DTT (dithiothreitol) to each sample to a final concentration of 10 mM, place it in a constant temperature mixer, and carry out the reduction reaction at 56°C for 30 minutes. After the reaction is completed, add IAA (iodoacetamide) to a final concentration of 40 mM, and carry out the alkylation reaction at room temperature and in the dark for 30 minutes.

[0084] (3) S-TRAP Protein Digestion: Add phosphate solution to the alkylated sample to a final concentration of 1.2%, and add binding buffer (90% methanol solution containing 100mM triethylammonium bicarbonate (TEAB)) equivalent to 7 times the lysis buffer. Place the sample in an S-trap column, centrifuge at 1,000g for 1 minute, wash the S-trap column three times with 400μL binding buffer, and add 0.4μg / μL Promega's trypsin (sequencing grade) to 125μL digestion buffer (50mM triethylammonium bicarbonate (TEAB)) at an enzyme-to-protein ratio of 1:25. Then incubate at 37°C for 16 hours.

[0085] (4) Elution and Reconstitution: Add 80 μL of 50 mM triethylammonium bicarbonate (TEAB) to the S-trap column after enzyme digestion, centrifuge at 1,000 g for 1 minute, and transfer the obtained peptide sample to a new tube; then add 80 μL of 0.2% formic acid aqueous solution to the S-trap column, centrifuge at 1,000 g for 1 minute, and collect the peptide sample. Add 80 μL of 50% acetonitrile (ACN) solution containing 0.2% formic acid to the S-trap column, centrifuge at 1,000 g for 1 minute, collect the peptide sample, and vacuum dry the collected peptide sample using a vacuum centrifuge concentrator. Reconstitute the dried sample with 0.1% trifluoroacetic acid (TFA) solution, and determine the peptide concentration using an ultra-micro spectrophotometer.

[0086] Example 2. Screening of biomarkers for the prediction or diagnosis of preeclampsia in early pregnancy To screen for biomarkers for the prediction or diagnosis of preeclampsia in early pregnancy, maternal plasma proteins were extracted in early pregnancy according to Example 1 and analyzed by LC / MS (liquid chromatography-mass spectrometry).

[0087] Specifically, mass spectrometric analysis was performed using a nano-level liquid chromatography-tandem mass spectrometry (Nano LC-MS / MS) system (Thermo Dionex Ultimate 3000 coupled with Thermo Orbitrap Exploris 480), according to the methods and conditions in Table 2 below.

[0088] Table 2

[0089] As part of LC-MS / MS data analysis, data independence acquisition (DIA) analysis was performed on the results, and database searches were conducted using DIA-NN software, which is specifically designed for DIA data analysis.

[0090] The search parameters are as follows.

[0091] - No library mode required (in the UniProt human protein database, set the peptide length to 7~50, set the number of missing cleavage sites to less than 1, and set cysteine ​​alkylation and methionine oxidation to fixed modification and variable modification, respectively).

[0092] - MBR (match-between-runs) mode.

[0093] For the search results, the false discovery rate (FDR) for both precursor ion and protein levels was controlled to be 1%. A hierarchical summary analysis based on precursor ion, peptide, and protein levels was performed using the median value, and the global distribution of all samples was normalized by quantiles at each level to identify blood proteins (i.e., characteristic markers) specifically overexpressed under each condition (normal and preeclampsia). For this purpose, protein characteristics meeting the following criteria (1) to (5) were defined as follows: When two groups are set up for comparison, (1) In each group, only proteins with a missing value of less than 50% are retained; (2) When the sample labels are randomly permuted 1,000 times, the log2 fold change falls within the top 15% of the generated empirical null hypothesis fold change distribution; (3) Empirical p-value < 0.05; (4) Median expression level > 0 under the target condition; and (5) Median expression level under other conditions < 0.

[0094] Based on the implementation results, proteins showing changes in expression will ultimately be screened as biomarkers for the prediction or diagnosis of preeclampsia in early pregnancy.

[0095] Figure 1 The results are from a heat map analysis of the levels of preeclampsia-specific biomarkers selected for prediction or diagnosis of preeclampsia in early pregnancy.

[0096] like Figure 1As shown, APOE, MMP2, LTA4H, ENG, GPT, SERPINB3, KRT9, CHI3L1, DSC2, SPP2, GFUS, CD109, SCARA5, MYOC, and ADA2 showed relatively higher levels in the preeclampsia group in early pregnancy compared to the control group. The levels of PSMD9, TREH, SERPINA7, TUBB, PFN1, HLA-C, G6PD, ANXA3, RAN, CTBS, GDF15, XYLT2, and GP6 were relatively low in the preeclampsia group during early pregnancy compared to the control group.

[0097] Example 3. Screening of biomarkers for the prediction or diagnosis of preeclampsia in mid-pregnancy To screen for biomarkers for the prediction or diagnosis of preeclampsia in the second trimester, maternal plasma proteins were extracted in the second trimester according to Example 1 and analyzed by mass spectrometry-based LC / MS.

[0098] The protein analysis was performed using the same method as in Example 2 above. Based on the results, proteins showing changes in expression were ultimately screened as biomarkers for the prediction or diagnosis of preeclampsia in the second trimester.

[0099] Figure 2 The results are from a heat map analysis of the levels of preeclampsia-specific biomarkers selected for prediction or diagnosis of preeclampsia in the second trimester.

[0100] like Figure 2 As shown, 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 preeclampsia group compared to the control group. ARID1B, WIPF1, CP, AGRN, CES2, TREH, APOB, SERPINE2, ITGA5, F7, HLA-C, GHR, LIPC, CAST, PRCP, ADAM17, FLNC, NOTUM, and VWA1 showed relatively low levels in the mid-pregnancy preeclampsia group compared to the control group.

[0101] Example 4. Screening of biomarkers for the diagnosis of preeclampsia in late pregnancy To screen for biomarkers for the diagnosis of preeclampsia in late pregnancy, maternal plasma proteins were extracted in late pregnancy according to Example 1 and analyzed by mass spectrometry-based LC / MS.

[0102] Protein analysis was performed using the same method as in Example 2 above. Based on the results, proteins showing changes in expression were ultimately screened as biomarkers for the diagnosis of preeclampsia in late pregnancy.

[0103] Figure 3 The results are from a heat map analysis of the levels of preeclampsia-specific biomarkers selected for diagnosis of preeclampsia in late pregnancy.

[0104] like Figure 3 As shown, 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-pregnancy preeclampsia group compared to the control group. The levels 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 were relatively low in the late-pregnancy preeclampsia group compared to the control group.

[0105] Based on the above description, those skilled in the art should understand that the present invention can be implemented in other specific forms without changing its technical concept or essential characteristics. In this regard, the experimental examples and embodiments described above should be understood in all respects as exemplary, not restrictive. The scope of the present invention is not defined by the foregoing description, but should be interpreted as including all changes or modifications derived from the meaning and scope of the following claims and their equivalents.

[0106] Industrial applicability This invention relates to disease-specific biomarkers for early prediction and diagnosis of preeclampsia and their uses, which can be used for early prediction and diagnosis of preeclampsia.

[0107] Related South Korean research and development projects - Project ID: 1465039723 Project Number: RS-2022-KH129953 - Name of the competent authority: Ministry of Health and Welfare - Name of the project management (professional) organization: Korea Health Industry Promotion Institute - Research Project Title: Cultivating Research-Oriented Hospitals - Research Project Title: Constructing a Platform for Addressing Unmet Medical Needs in Pregnancy and Childbirth Based on Future Innovative Technologies - Name of the institution implementing the research project: Pentangche Hospital - Research period for the current year: January 1, 2023 to December 31, 2023.

Claims

1. A biomarker composition for the prediction or diagnosis of preeclampsia in early pregnancy, comprising one or more proteins or genes encoding the 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.

2. A composition for the prediction or diagnosis of preeclampsia in early pregnancy, comprising a preparation for measuring the mRNA level of one or more proteins or genes encoding the 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.

3. The composition for predicting or diagnosing preeclampsia in early pregnancy according to claim 2, wherein, The formulation for measuring the protein level comprises one or more selected from the group consisting of antibodies, interacting proteins, oligopeptides, ligands, nanoparticles, and aptamers that specifically bind to the protein or its peptide fragments.

4. The composition for predicting or diagnosing preeclampsia in early pregnancy according to claim 2, wherein, The preparation for determining the mRNA level of the gene comprises one or more selected from the group consisting of primers, probes, peptide nucleic acids, and antisense nucleotides that specifically bind to the gene.

5. A kit for predicting or diagnosing preeclampsia in early pregnancy, comprising the composition of any one of claims 2 to 4.

6. The kit for predicting or diagnosing preeclampsia in early pregnancy according to claim 5, wherein, The kit is an enzyme-linked immunosorbent assay kit, a protein chip kit, a rapid detection kit, a multiple reaction monitoring kit, a reverse transcription polymerase chain reaction kit, or a DNA chip kit.

7. A method for providing information for the prediction or diagnosis of preeclampsia in early pregnancy, comprising: The steps include determining the mRNA level of one or more proteins or genes encoding the 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 in samples isolated from subjects in early pregnancy; and comparing the mRNA level of the proteins or genes encoding the proteins with that of a normal control group in early pregnancy.

8. The method for providing information for predicting or diagnosing preeclampsia in early pregnancy according to claim 7, wherein, The steps for determining protein levels are performed by selecting one or more methods from the group consisting of protein mass spectrometry, protein chip analysis, immunoassay, ligand binding analysis, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, surface-enhanced laser desorption / ionization time-of-flight mass spectrometry, radioimmunoassay, radioimmunodiffusion, Octelloni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis, liquid chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, protein immunoblotting, and enzyme-linked immunosorbent assay.

9. The method for providing information for predicting or diagnosing preeclampsia in early pregnancy according to claim 7, wherein, The step of determining the mRNA level of the gene is performed by selecting one or more of the following methods: reverse transcription polymerase chain reaction, competitive reverse transcription polymerase chain reaction, real-time reverse transcription polymerase chain reaction, ribonuclease protection assay, RNA blotting, and DNA microarray.

10. The method for providing information for predicting or diagnosing preeclampsia in early pregnancy according to claim 7, wherein, Also includes: Preeclampsia is defined as an increase in the mRNA level of one or more proteins or genes encoding the proteins selected from the group consisting of APOE, MMP2, LTA4H, ENG, GPT, SERPINB3, KRT9, CHI3L1, DSC2, SPP2, GFUS, CD109, SCARA5, MYOC, and ADA2 compared to the control group. Alternatively, a step is taken to determine preeclampsia when the mRNA level of one or more proteins or genes encoding such proteins selected from the group consisting of PSMD9, TREH, SERPINA7, TUBB, PFN1, HLA-C, G6PD, ANXA3, RAN, CTBS, GDF15, XYLT2, and GP6 is lower than that of the control group.

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