Disease-specific biomarker for predicting second-trimester preterm birth and use thereof

Abstract

The present invention relates to a disease-specific biomarker for early prediction and diagnosis of preterm birth, and a use thereof. According to one aspect of the present invention, a composition for predicting preterm birth or a method for providing information for prediction of preterm birth can simply and effectively predict and diagnose a disease by measuring and comparing disease-specific proteins changing in a patient or the levels of mRNA of genes encoding the disease-specific proteins.

Description

Disease-specific biomarkers for predicting preterm birth in the second trimester and their use

[0001] It relates to disease-specific biomarkers and their uses for early prediction and diagnosis of preterm birth.

[0002] Preterm birth refers to delivery occurring between 20 weeks and 0 days and 36 weeks and 6 days of gestation. Preterm birth accounts for approximately 6 to 15% of all deliveries and is responsible for 80% of neonatal deaths. Therefore, early prediction and diagnosis of preterm birth are crucial for preventing preterm birth and improving prognosis through neonatal treatment.

[0003] To predict premature birth, an ultrasound examination using cervical length measurement after 16 weeks of pregnancy is used, but the sensitivity is 27.8% for ≤ 25 mm and 47.2% for ≤ 30 mm, so the accuracy is very low (Journal of the Korean Society of Obstetrics and Gynecology, 2000, Seong Seok-ju et al.).

[0004] Accordingly, there is a need for a diagnostic test that can effectively predict preterm birth and simplify diagnosis, but there is currently no conventional technology using biomarkers developed.

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

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

[0007] Another aspect provides for the use of biomarkers comprising proteins or genes encoding them for the prediction of preterm birth in early, mid or late pregnancy.

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

[0009] 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 of preterm birth in early, middle or late pregnancy.

[0010] Another aspect provides a use for preparing a composition for predicting preterm birth 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 preterm birth in early, mid or late pregnancy, comprising the composition for predicting preterm birth.

[0012] Another aspect provides a method for providing information for predicting preterm birth 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 preterm birth 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 early, mid or late preterm birth, comprising a protein or a gene encoding the same.

[0015] Another aspect provides for the use of biomarkers comprising proteins or genes encoding them for the prediction of preterm birth in early, mid or late pregnancy.

[0016] Another aspect provides a composition for predicting preterm birth in early, mid or late pregnancy, comprising a preparation measuring the level of mRNA of the protein or a gene encoding the protein.

[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 of preterm birth in early, middle or late pregnancy.

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

[0019] In a biomarker composition for predicting premature birth in early pregnancy or a composition for predicting premature birth in early pregnancy according to one specific example, the protein is IGHV3-72, IGHV3OR16-9, ARID1B, IGLC2, IGLC3, IGLC6, IGLC7, GSK3A, NECTIN2, ENTPD5, A2M, IGHA1, APOA1, SLC4A1, FN1, APOB, VWF, P4HB, CAPN1, MMP2, ALDOC, SELL, AOC1, MST1, SERPINB3, CASP14, THBS4, FLT4, LRP1, SPP2, SCARA5, MUC15, MYOC, EMILIN2, SEMA4A, TNS1, PCYOX1, HEG1, SERPINA3, CGB1, CGB2, CGB3, CGB7, RNASET2, F2, FGB, It may include one or more proteins selected from the group consisting of FGG, LCAT, SERPINA5, SERPINA7, ANXA6, CPN2, TALDO1, RAN, MERTK, SMPDL3A, GP6, and CTSZ.

[0020] In a biomarker composition for predicting mid-trimester preterm birth or a composition for predicting mid-trimester preterm birth according to one specific example, the protein may include one or more proteins selected from the group consisting of F12, FN1, VWF, HAPLN1, PTPRB, KRT9, GP5, PSMB3, SEMA3B, KRT80, SPON2, UGGT1, PSG8, CES2, FGA, C4BPA, DCN, HMBS, SRGN, GHR, ACTN1, SMPD1, CAST, FCGRT, NUTF2, GFUS, ITIH5, PRAP1, MYOC, and GLOD4.

[0021] In a biomarker composition for predicting late-stage premature birth according to one specific example or a composition for predicting late-stage premature birth, the protein is WIPF1, ALDH1A1, PNP, HBD, VTN, CAT, GAPDH, APOA4, C4B, RPS27A, UBA52, UBB, UBC, KIT, ESD, ALAD, GOT1, TCN1, PSMB1, CAST, PRDX6, BLVRB, GDI2, APLP1, BLVRA, VCP, ACTB, ACTG1, PSMA6, RAN, SELENBP1, GFUS, TSKU, PARK7, VN1R1, VN1R3, PCDH18, C1S, CLSTN1, OLFM2, AHSG, DCN, UMOD, ACE, SMPD1, VCAM1, IGFBP5, CALR, MCAM, LUM, NAGLU, CDH6, It may include one or more proteins selected from the group consisting of SOS1, DDR1, PTK7, LSAMP, PON3, PRSS8, FAT4, SCARA5, NEGR1, CLEC14A, OAF, CILP2, SMPDL3A, NEO1, TWSG1, CD93, and SMARCAL1.

[0022] In this specification, the term “preterm birth” is also referred to as “early delivery” and refers to delivery that occurs between 20 weeks and 0 days and 36 weeks and 6 days of gestation.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] The term “level measurement” herein may include measuring the degree of expression or activity of a marker for preterm birth in a biological sample to predict or diagnose preterm birth.

[0027] Specifically, measuring the level of the protein may mean measuring the degree of expression of the protein that changes according to the risk or onset of premature birth in a pregnant woman in the early, middle or late pregnancy.

[0028] Specifically, measuring the level of the mRNA may mean measuring the degree of expression of the mRNA that changes according to the risk or onset of premature birth in a pregnant woman in the early, middle or late pregnancy.

[0029]

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036]

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043]

[0044] Another aspect provides a kit for predicting preterm birth in early, mid or late pregnancy, comprising the composition for predicting preterm birth.

[0045] 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.

[0046] The kit may further comprise one or more other component compositions, solutions or devices suitable for the analytical method.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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)).

[0054] 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 of premature birth through the algorithm.

[0055]

[0056] Another aspect provides a method for providing information for predicting preterm birth 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.

[0057] Another aspect provides a method for predicting preterm birth 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.

[0058] 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.

[0059] 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 premature birth, has developed premature birth or is suspected of developing premature birth, and may be at least one selected from the group consisting of whole blood, leukocytes, peripheral blood mononuclear cells, leukocyte buffy coat, blood including 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 a liquid biopsy, for example, blood, serum or plasma.

[0060] 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.

[0061] In a method for providing information for predicting preterm birth in early pregnancy according to one specific example, the protein is IGHV3-72, IGHV3OR16-9, ARID1B, IGLC2, IGLC3, IGLC6, IGLC7, GSK3A, NECTIN2, ENTPD5, A2M, IGHA1, APOA1, SLC4A1, FN1, APOB, VWF, P4HB, CAPN1, MMP2, ALDOC, SELL, AOC1, MST1, SERPINB3, CASP14, THBS4, FLT4, LRP1, SPP2, SCARA5, MUC15, MYOC, EMILIN2, SEMA4A, TNS1, PCYOX1, HEG1, SERPINA3, CGB1, CGB2, CGB3, CGB7, RNASET2, F2, FGB, FGG, LCAT, It may include one or more proteins selected from the group consisting of SERPINA5, SERPINA7, ANXA6, CPN2, TALDO1, RAN, MERTK, SMPDL3A, GP6, and CTSZ.

[0062] In a method for providing information for predicting preterm birth in the second trimester of pregnancy according to one specific example, the protein may include one or more proteins selected from the group consisting of F12, FN1, VWF, HAPLN1, PTPRB, KRT9, GP5, PSMB3, SEMA3B, KRT80, SPON2, UGGT1, PSG8, CES2, FGA, C4BPA, DCN, HMBS, SRGN, GHR, ACTN1, SMPD1, CAST, FCGRT, NUTF2, GFUS, ITIH5, PRAP1, MYOC and GLOD4.

[0063] In a method for providing information for predicting preterm birth in late pregnancy according to one specific example, the protein is WIPF1, ALDH1A1, PNP, HBD, VTN, CAT, GAPDH, APOA4, C4B, RPS27A, UBA52, UBB, UBC, KIT, ESD, ALAD, GOT1, TCN1, PSMB1, CAST, PRDX6, BLVRB, GDI2, APLP1, BLVRA, VCP, ACTB, ACTG1, PSMA6, RAN, SELENBP1, GFUS, TSKU, PARK7, VN1R1, VN1R3, PCDH18, C1S, CLSTN1, OLFM2, AHSG, DCN, UMOD, ACE, SMPD1, VCAM1, IGFBP5, CALR, MCAM, LUM, NAGLU, CDH6, SOS1, DDR1, PTK7, It may include one or more proteins selected from the group consisting of LSAMP, PON3, PRSS8, FAT4, SCARA5, NEGR1, CLEC14A, OAF, CILP2, SMPDL3A, NEO1, TWSG1, CD93, and SMARCAL1.

[0064] 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).

[0065] 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.

[0066] In one specific example, the method for providing information for predicting premature birth in early pregnancy comprises predicting that there is a risk of premature birth when the level of mRNA of one or more proteins selected from the group consisting of IGHV3-72, IGHV3OR16-9, ARID1B, IGLC2, IGLC3, IGLC6, IGLC7, GSK3A, NECTIN2, ENTPD5, A2M, IGHA1, APOA1, SLC4A1, FN1, APOB, VWF, P4HB, CAPN1, MMP2, ALDOC, SELL, AOC1, MST1, SERPINB3, CASP14, THBS4, FLT4, LRP1, SPP2, SCARA5, MUC15, MYOC, EMILIN2, SEMA4A, TNS1, PCYOX1 and HEG1 or a gene encoding the same is increased compared to a control group, or SERPINA3, The method may further include a step of predicting that there is a risk of premature birth when the level of mRNA of one or more proteins selected from the group consisting of CGB1, CGB2, CGB3, CGB7, RNASET2, F2, FGB, FGG, LCAT, SERPINA5, SERPINA7, ANXA6, CPN2, TALDO1, RAN, MERTK, SMPDL3A, GP6, and CTSZ or a gene encoding the same is decreased compared to the control group.

[0067] In one specific example, the method for providing information for predicting preterm birth in the second trimester may further include a step of predicting that there is a risk of preterm birth when the level of mRNA of one or more proteins selected from the group consisting of F12, FN1, VWF, HAPLN1, PTPRB, KRT9, GP5, PSMB3, SEMA3B, KRT80, SPON2, UGGT1, and PSG8 or a gene encoding the same is increased compared to a control group, or a step of predicting that there is a risk of preterm birth when the level of mRNA of one or more proteins selected from the group consisting of CES2, FGA, C4BPA, DCN, HMBS, SRGN, GHR, ACTN1, SMPD1, CAST, FCGRT, NUTF2, GFUS, ITIH5, PRAP1, MYOC, and GLOD4 or a gene encoding the same is decreased compared to a control group.

[0068] In one specific example, the method for providing information for predicting premature birth in the late stage of pregnancy comprises predicting that there is a risk of premature birth when the level of mRNA of one or more proteins selected from the group consisting of WIPF1, ALDH1A1, PNP, HBD, VTN, CAT, GAPDH, APOA4, C4B, RPS27A, UBA52, UBB, UBC, KIT, ESD, ALAD, GOT1, TCN1, PSMB1, CAST, PRDX6, BLVRB, GDI2, APLP1, BLVRA, VCP, ACTB, ACTG1, PSMA6, RAN, SELENBP1, GFUS, TSKU, PARK7, VN1R1, VN1R3 and PCDH18 or a gene encoding the same is increased compared to a control group, or predicting that there is a risk of premature birth when the level of mRNA of the gene encoding the protein is increased compared to a control group, or predicting that there is a risk of premature birth when the level of mRNA of the gene encoding the protein is increased compared to a control group, or predicting that there is a risk of premature birth when the level of mRNA of the gene encoding the protein is increased compared to a control group, or predicting that there is a risk of premature birth when the level of the mRNA of the gene encoding the protein is increased compared to a control group, or predicting that there is a risk of premature birth when the level of the mRNA of the gene encoding the gene ... encoding the gene encoding the gene encoding the gene encoding the gene encoding the gene encoding the gene encoding the gene encoding the gene encoding the gene encoding the gene encoding the gene encoding the gene encoding the gene The method may further include a step of predicting that there is a risk of premature birth when the level of mRNA of one or more proteins selected from the group consisting of IGFBP5, CALR, MCAM, LUM, NAGLU, CDH6, SOS1, DDR1, PTK7, LSAMP, PON3, PRSS8, FAT4, SCARA5, NEGR1, CLEC14A, OAF, CILP2, SMPDL3A, NEO1, TWSG1, CD93, and SMARCAL1 or a gene encoding the same is decreased compared to the control group.

[0069] 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.

[0070] 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.

[0071] In one specific example, the method of providing information for predicting premature birth can be performed using a plurality of panels.

[0072] A composition for predicting premature birth according to a certain aspect or a method for providing information for predicting the same can simply and effectively predict and diagnose premature birth 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.

[0073] Figure 1 shows the results of a heat map analysis of the levels of early pregnancy preterm birth-specific biomarkers selected for the prediction of tentative early preterm birth.

[0074] Figure 2 shows the results of a heat map analysis of the levels of mid-trimester preterm birth-specific biomarkers selected for the prediction of mid-trimester preterm birth.

[0075] Figure 3 shows the results of a heat map analysis of the levels of specific biomarkers for late-term preterm birth selected for the prediction of late-term preterm birth.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] Throughout the specification of the present invention, “A and / or B” means A or B, or A and B.

[0080]

[0081] Example 1. Extraction of plasma proteins from early, mid, and late pregnancy maternal blood.

[0082] Plasma proteins were extracted from maternal plasma samples from 10 normal pregnant women in the first, second, and third trimesters of pregnancy, from 10 preterm pregnant women in the first and second trimesters of pregnancy, and from maternal plasma samples from 5 preterm pregnant women in the third trimester of pregnancy.

[0083]

[0084] 1.1 Plasma depletion

[0085] 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.

[0086]

[0087] 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-

[0088]

[0089] 1.2 Protein lysis and digestion

[0090] Protein dissolution and digestion are performed in the following steps (1) to (4).

[0091] (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.

[0092] (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.

[0093] (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.

[0094] (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.

[0095]

[0096] Example 2. Selection of biomarkers for predicting preterm birth in early pregnancy.

[0097] To select a biomarker for predicting preterm birth 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.

[0098] 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.

[0099]

[0100] LC & LC-MS / MS systemNano-LC Ultimate 3000 & Thermo Orbitrap Exploris 480ColumnTrap column: Acclaim PepMapTM 100, 100 ㎛ x 2 ㎝Analytical column: PepMap TM RSLC C18, 75 ㎛ (%)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 %

[0101]

[0102] 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.

[0103]

[0104] Search parameters are as follows:

[0105] - 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)

[0106] - MBR (match-between-runs) mode.

[0107]

[0108] 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 preterm birth), protein signatures satisfying the following criteria (1) to (5) were defined:

[0109] When placed into two groups for comparison,

[0110] (1) In each group, only proteins with missing value <= 50% are retained.

[0111] (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.

[0112] (3) empirical p-value <0.05,

[0113] (4) median expression in the condition of interest >0, and

[0114] (5) median expression in the other conditions <0.

[0115]

[0116] As a result of the performance, the protein showing the final expression change was selected as a biomarker for predicting preterm birth in early pregnancy.

[0117]

[0118] Figure 1 shows the results of a heat map analysis of the levels of early pregnancy preterm birth-specific biomarkers selected for provisional early preterm birth prediction.

[0119] As shown in Figure 1, IGHV3-72, IGHV3OR16-9, ARID1B, IGLC2, IGLC3, IGLC6, IGLC7, GSK3A, NECTIN2, ENTPD5, A2M, IGHA1, APOA1, SLC4A1, FN1, APOB, VWF, P4HB, CAPN1, MMP2, ALDOC, SELL, AOC1, MST1, SERPINB3, CASP14, THBS4, FLT4, LRP1, SPP2, SCARA5, MUC15, MYOC, EMILIN2, SEMA4A, TNS1, PCYOX1, and HEG1 showed relatively higher levels in the early preterm birth group compared to the control group.

[0120] For SERPINA3, CGB1, CGB2, CGB3, CGB7, RNASET2, F2, FGB, FGG, LCAT, SERPINA5, SERPINA7, ANXA6, CPN2, TALDO1, RAN, MERTK, SMPDL3A, GP6, and CTSZ, relatively lower levels were observed in the preterm birth group in early pregnancy compared to the control group.

[0121]

[0122] Example 3. Selection of biomarkers for predicting preterm birth in the second trimester of pregnancy.

[0123] To select a biomarker for predicting mid-trimester preterm birth, analysis was performed using mass spectrometry-based LC / MS after protein extraction from mid-trimester maternal plasma according to Example 1.

[0124] The protein analysis was performed in the same manner as in Example 2 above. As a result, the protein showing the final expression change was selected as a biomarker for predicting mid-term premature birth.

[0125]

[0126] Figure 2 shows the results of a heat map analysis of the levels of mid-trimester preterm birth-specific biomarkers selected for provisional mid-trimester preterm birth prediction.

[0127] As shown in Figure 2, for F12, FN1, VWF, HAPLN1, PTPRB, KRT9, GP5, PSMB3, SEMA3B, KRT80, SPON2, UGGT1, and PSG8, the levels were relatively higher in the mid-preterm pregnancy group compared to the control group.

[0128] For CES2, FGA, C4BPA, DCN, HMBS, SRGN, GHR, ACTN1, SMPD1, CAST, FCGRT, NUTF2, GFUS, ITIH5, PRAP1, MYOC, and GLOD4, relatively lower levels were observed in the mid-preterm pregnancy group compared to the control group.

[0129]

[0130] Example 4. Selection of biomarkers for predicting preterm birth in late pregnancy.

[0131] To select a biomarker for predicting preterm birth 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.

[0132] The protein analysis was performed in the same manner as in Example 2. As a result, the protein showing the final expression change was selected as a biomarker for predicting premature birth in late pregnancy.

[0133]

[0134] Figure 3 shows the results of a heat map analysis of the levels of specific biomarkers for late-term preterm birth selected for provisional prediction of late-term preterm birth.

[0135] As shown in Figure 3, WIPF1, ALDH1A1, PNP, HBD, VTN, CAT, GAPDH, APOA4, C4B, RPS27A, UBA52, UBB, UBC, KIT, ESD, ALAD, GOT1, TCN1, PSMB1, CAST, PRDX6, BLVRB, GDI2, APLP1, BLVRA, VCP, ACTB, ACTG1, PSMA6, RAN, SELENBP1, GFUS, TSKU, PARK7, VN1R1, VN1R3 and PCDH18 showed relatively higher levels in the late pregnancy preterm birth group compared to the control group.

[0136] For C1S, CLSTN1, OLFM2, AHSG, DCN, UMOD, ACE, SMPD1, VCAM1, IGFBP5, CALR, MCAM, LUM, NAGLU, CDH6, SOS1, DDR1, PTK7, LSAMP, PON3, PRSS8, FAT4, SCARA5, NEGR1, CLEC14A, OAF, CILP2, SMPDL3A, NEO1, TWSG1, CD93, and SMARCAL1, relatively lower levels were observed in the late-term preterm birth group compared to the control group.

[0137]

[0138] 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.

[0139]

[0140] The present invention relates to a disease-specific biomarker for early prediction and diagnosis of premature birth and its use, which can be used for early prediction and diagnosis of premature birth.

[0141] Related national research and development projects

[0142] - Assignment ID: 1465039723

[0143] - Assignment Number: RS-2022-KH129953

[0144] - Ministry name: Ministry of Health and Welfare

[0145] - Project Management (Professional) Organization Name: Korea Health Industry Development Institute

[0146] - Research Project Name: Research-Oriented Hospital Development

[0147] Research Project Name: Establishing a Platform to Address Unmet Pregnancy and Childbirth Medical Needs Based on Future Innovation Technology

[0148] - Project implementation organization name: Bundang Cha Hospital

[0149] - Research period for the current year: January 1, 2023 - December 31, 2023

Claims

1. A biomarker composition for predicting mid-trimester birth, comprising one or more proteins selected from the group consisting of F12, FN1, VWF, HAPLN1, PTPRB, KRT9, GP5, PSMB3, SEMA3B, KRT80, SPON2, UGGT1, PSG8, CES2, FGA, C4BPA, DCN, HMBS, SRGN, GHR, ACTN1, SMPD1, CAST, FCGRT, NUTF2, GFUS, ITIH5, PRAP1, MYOC and GLOD4, or a gene encoding the same. A composition for predicting preterm birth in the second trimester, comprising a preparation for measuring the level of mRNA of one or more proteins selected from the group consisting of 2.F12, FN1, VWF, HAPLN1, PTPRB, KRT9, GP5, PSMB3, SEMA3B, KRT80, SPON2, UGGT1, PSG8, CES2, FGA, C4BPA, DCN, HMBS, SRGN, GHR, ACTN1, SMPD1, CAST, FCGRT, NUTF2, GFUS, ITIH5, PRAP1, MYOC and GLOD4.

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

5. A kit for predicting preterm birth in the second trimester, comprising a composition according to any one of claims 2 to 4.

6. A kit for predicting mid-trimester premature birth 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 F12, FN1, VWF, HAPLN1, PTPRB, KRT9, GP5, PSMB3, SEMA3B, KRT80, SPON2, UGGT1, PSG8, CES2, FGA, C4BPA, DCN, HMBS, SRGN, GHR, ACTN1, SMPD1, CAST, FCGRT, NUTF2, GFUS, ITIH5, PRAP1, MYOC and GLOD4 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 preterm birth in the second trimester.

8. In claim 7, the step of measuring the level of the protein comprises: A method for providing information for predicting preterm birth in the second trimester, 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, Oukteroni 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 preterm birth in the second trimester, wherein the method is 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 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 F12, FN1, VWF, HAPLN1, PTPRB, KRT9, GP5, PSMB3, SEMA3B, KRT80, SPON2, UGGT1 and PSG8 or a gene encoding the same is increased compared to the control group, it is predicted that there is a risk of premature birth, or A method for predicting a risk of premature birth further comprising a step of predicting that there is a risk of premature birth when the level of mRNA of one or more proteins selected from the group consisting of CES2, FGA, C4BPA, DCN, HMBS, SRGN, GHR, ACTN1, SMPD1, CAST, FCGRT, NUTF2, GFUS, ITIH5, PRAP1, MYOC and GLOD4 or a gene encoding the same is decreased compared to the control group. A method for providing information for predicting preterm birth in the second trimester.

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