Disease-specific biomarker for predicting and diagnosing mid-pregnancy gestational diabetes, and use thereof

Abstract

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

Description

Disease-specific biomarkers for predicting and diagnosing gestational diabetes in the second trimester and their uses

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

[0002] Gestational diabetes is a disease in which diabetes is first discovered after the 20th week of pregnancy in a woman who did not have diabetes. The prevalence rate varies by country and race, but in Korea, it accounts for more than 10% of all pregnant women and the prevalence rate is increasing every year.

[0003] Diagnosis of gestational diabetes is typically conducted in two stages: screening and diagnostic testing. In the screening test (stage 1), a woman consumes 50 grams of sugar between 24 and 28 weeks of pregnancy, regardless of the time of the test or whether she ate anything. A positive result is a plasma glucose concentration of 140 mg / dL or higher (130 mg / dL for high-risk mothers) one hour later. In this case, a 100-gram oral glucose tolerance test (diagnostic test) is performed. In the diagnostic test (stage 2), a woman consumes 100 grams of sugar in the morning after fasting for at least eight hours or an overnight fast. If two or more of the following four criteria are met, gestational diabetes is diagnosed:

[0004] Fasting plasma glucose ≥ 95 mg / dL

[0005] Plasma blood glucose ≥ 180 mg / dL 1 hour after glucose load

[0006] Plasma blood glucose ≥ 155 mg / dL 2 hours after glucose load

[0007] Plasma blood glucose ≥ 140 mg / dL 3 hours after glucose load.

[0008] The therapeutic approach to gestational diabetes is to manage blood sugar levels to reach target levels through diet and exercise. If target blood sugar levels are difficult to achieve with diet and exercise, insulin treatment is administered.

[0009] Gestational diabetes increases the risk of macrosomia, which can lead to difficult labor at birth, and increases the risk of complications for the mother and newborn during labor, as well as the risk of developing neonatal hypoglycemia, hypocalcemia, and neonatal respiratory distress syndrome. In addition, there is a risk of gestational hypertension and premature birth, and in the long term, the chance of gestational diabetes recurring in the next pregnancy is more than 50%. Furthermore, women who have had gestational diabetes are at high risk of developing diabetes, and children born to mothers with gestational diabetes are about twice as likely to develop childhood obesity and metabolic syndrome as children born to mothers without gestational diabetes.

[0010] Accordingly, in order to prevent gestational diabetes and improve the prognosis through treatment during the disease, a testing method that can effectively predict gestational diabetes 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.

[0011] Accordingly, the inventors of the present invention have discovered the world's first gestational diabetes-specific biomarker that shows significant changes in patients, and have developed a simple and effective method for early prediction and diagnosis of gestational diabetes.

[0012] One aspect is to provide a biomarker composition for predicting or diagnosing early, mid or late pregnancy gestational diabetes, comprising a protein or a gene encoding the same.

[0013] Another aspect provides the use of a biomarker comprising a protein or a gene encoding the same for the prediction or diagnosis of gestational diabetes in early, mid or late pregnancy.

[0014] Another aspect provides a composition for predicting or diagnosing gestational diabetes in early, middle or late pregnancy, comprising a preparation for measuring the level of mRNA of the protein or the gene encoding it.

[0015] Another aspect provides a use of a formulation for measuring the level of mRNA of the protein or the gene encoding it for predicting or diagnosing gestational diabetes in early, middle or late pregnancy.

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

[0017] Another aspect is to provide a kit for predicting or diagnosing gestational diabetes in early, middle or late pregnancy, comprising the composition for predicting or diagnosing gestational diabetes.

[0018] Another aspect provides a method for providing information for predicting or diagnosing gestational diabetes 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.

[0019] Another aspect provides a method for predicting or diagnosing gestational diabetes 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.

[0020] One aspect provides a biomarker composition for predicting or diagnosing gestational diabetes in early, mid or late pregnancy, comprising a protein or a gene encoding the same.

[0021] Another aspect provides the use of biomarkers comprising proteins or genes encoding them for the prediction or diagnosis of gestational diabetes in early, mid or late pregnancy.

[0022] Another aspect provides a composition for predicting or diagnosing gestational diabetes in early, middle or late pregnancy, comprising a preparation for measuring the level of mRNA of the protein or the gene encoding it.

[0023] Another aspect provides a use of a formulation for measuring the level of mRNA of the protein or the gene encoding it for predicting or diagnosing gestational diabetes in early, middle or late pregnancy.

[0024] Another aspect provides a use for preparing a composition for predicting or diagnosing gestational diabetes in early, middle or late pregnancy, comprising a preparation measuring the level of mRNA of the protein or the gene encoding it.

[0025] In a biomarker composition for predicting or diagnosing gestational diabetes in early pregnancy according to one specific example or a composition for predicting or diagnosing gestational diabetes in early pregnancy, the protein may include one or more proteins selected from the group consisting of GSK3A, FCGR3B, C3, LDLR, FN1, P4HB, TUBB, COL1A2, SELL, GPT, SERPINB3, DDT, ERAP2, PRAP1, PAPPA2, DPP3, FBLN1, SDCBP, TREH, SERPINE2, UMOD, SLC3A2, HLA-C, LIPC, PTPRD, RAN, CTBS, GALNT1, DSG2, NECTIN1 and GDF15.

[0026] In a biomarker composition for predicting or diagnosing mid-trimester gestational diabetes according to one specific example or a composition for predicting or diagnosing mid-trimester gestational diabetes, the protein may include one or more proteins selected from the group consisting of IGHA1, CRP, LTF, GPI, RET, COL1A2, GSTP1, MMP9, TYMP, PTPRB, RNASE4, CAMP, PPIA, ENPEP, DSC3, ERAP2, MEGF8, OIT3, TSEN34, CHGA, CR2, CAST, GFUS, FLNC, VASN, FERMT3, KCTD12, PRAP1, PGLYRP2, ERVMER34-1, SLC38A10, MYRF and RFNG.

[0027] In a biomarker composition for diagnosing late-stage gestational diabetes according to one specific example or a composition for diagnosing late-stage gestational diabetes, the protein may include one or more proteins selected from the group consisting of SVOPL, APOM, KLKB1, SOD2, PSG1, COL6A2, TCN1, PTPRM, CHI3L1, B2M, LTBP1, NECTIN2, RNASET2, ISLR, CUTA, LILRB5, SOD1, HLA-C, PTPRD, CALR, S100A7, CDH5, CACNA2D1, PTPRS, NEGR1 and SMPDL3A.

[0028] As used herein, the term “gestational diabetes” means diabetes that is first detected after the 20th week of pregnancy in a woman who did not have diabetes.

[0029] 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.

[0030] 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.

[0031] Previously, gestational diabetes could only be diagnosed after 20 weeks of pregnancy, but according to the composition for predicting or diagnosing gestational diabetes or the method for providing information for predicting or diagnosing the same according to the present invention, gestational diabetes 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.

[0032] 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.

[0033] The term “level measurement” herein may include measuring the degree of expression or activity of a marker for gestational diabetes in a biological sample to predict or diagnose gestational diabetes.

[0034] Specifically, measuring the level of the protein may mean measuring the degree of expression of the protein that changes with the onset of gestational diabetes in a pregnant woman in the early, middle or late trimester of pregnancy.

[0035] Specifically, measuring the level of the mRNA may mean measuring the degree of expression of the mRNA that changes according to the onset of gestational diabetes in a pregnant woman in the early, middle or late trimester of pregnancy.

[0036]

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043]

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050]

[0051] Another aspect provides a kit for predicting or diagnosing gestational diabetes in early, mid or late pregnancy, comprising the composition for predicting or diagnosing gestational diabetes.

[0052] 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.

[0053] The kit may further comprise one or more other component compositions, solutions or devices suitable for the analytical method.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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)).

[0061] 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 prediction or diagnosis of gestational diabetes through the algorithm.

[0062]

[0063] Another aspect provides a method for providing information for predicting or diagnosing gestational diabetes 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.

[0064] Another aspect provides a method for predicting or diagnosing gestational diabetes 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.

[0065] 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.

[0066] The term “sample” as used herein means any material, biological fluid, tissue or cell obtained from or derived from a subject who has developed gestational diabetes or is suspected of developing gestational diabetes, 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.

[0067] 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.

[0068] In a method for providing information for predicting or diagnosing gestational diabetes in early pregnancy according to one specific example, the protein may include one or more proteins selected from the group consisting of GSK3A, FCGR3B, C3, LDLR, FN1, P4HB, TUBB, COL1A2, SELL, GPT, SERPINB3, DDT, ERAP2, PRAP1, PAPPA2, DPP3, FBLN1, SDCBP, TREH, SERPINE2, UMOD, SLC3A2, HLA-C, LIPC, PTPRD, RAN, CTBS, GALNT1, DSG2, NECTIN1 and GDF15.

[0069] In a method for providing information for predicting or diagnosing mid-trimester gestational diabetes according to one specific example, the protein may include one or more proteins selected from the group consisting of IGHA1, CRP, LTF, GPI, RET, COL1A2, GSTP1, MMP9, TYMP, PTPRB, RNASE4, CAMP, PPIA, ENPEP, DSC3, ERAP2, MEGF8, OIT3, TSEN34, CHGA, CR2, CAST, GFUS, FLNC, VASN, FERMT3, KCTD12, PRAP1, PGLYRP2, ERVMER34-1, SLC38A10, MYRF and RFNG.

[0070] In a method for providing information for diagnosing gestational diabetes in late pregnancy according to one specific example, the protein may include one or more proteins selected from the group consisting of SVOPL, APOM, KLKB1, SOD2, PSG1, COL6A2, TCN1, PTPRM, CHI3L1, B2M, LTBP1, NECTIN2, RNASET2, ISLR, CUTA, LILRB5, SOD1, HLA-C, PTPRD, CALR, S100A7, CDH5, CACNA2D1, PTPRS, NEGR1 and SMPDL3A.

[0071] 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).

[0072] 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.

[0073] In one specific example, the method for providing information for predicting or diagnosing gestational diabetes in early pregnancy may further include a step of determining gestational diabetes when the level of mRNA of one or more proteins selected from the group consisting of GSK3A, FCGR3B, C3, LDLR, FN1, P4HB, TUBB, COL1A2, SELL, GPT, SERPINB3, DDT, ERAP2, PRAP1, PAPPA2, and DPP3 or a gene encoding the same is increased compared to a control group, or determining gestational diabetes when the level of mRNA of one or more proteins selected from the group consisting of FBLN1, SDCBP, TREH, SERPINE2, UMOD, SLC3A2, HLA-C, LIPC, PTPRD, RAN, CTBS, GALNT1, DSG2, NECTIN1, and GDF15 or a gene encoding the same is decreased compared to a control group.

[0074] In one specific example, the method for providing information for predicting or diagnosing mid-trimester gestational diabetes may further include a step of determining gestational diabetes when the level of mRNA of one or more proteins selected from the group consisting of IGHA1, CRP, LTF, GPI, RET, COL1A2, GSTP1, MMP9, TYMP, PTPRB, RNASE4, CAMP, PPIA, ENPEP, DSC3, ERAP2, MEGF8, OIT3, and TSEN34 or a gene encoding the same is increased compared to a control group, or when the level of mRNA of one or more proteins selected from the group consisting of CHGA, CR2, CAST, GFUS, FLNC, VASN, FERMT3, KCTD12, PRAP1, PGLYRP2, ERVMER34-1, SLC38A10, MYRF, and RFNG or a gene encoding the same is decreased compared to a control group.

[0075] In one specific example, the method for providing information for diagnosing gestational diabetes in the late stage of pregnancy may further include a step of determining gestational diabetes when the level of mRNA of one or more proteins selected from the group consisting of SVOPL, APOM, KLKB1, SOD2, PSG1, COL6A2, TCN1, PTPRM, CHI3L1, B2M and LTBP1 or a gene encoding the same is increased compared to a control group, or determining gestational diabetes when the level of mRNA of one or more proteins selected from the group consisting of NECTIN2, RNASET2, ISLR, CUTA, LILRB5, SOD1, HLA-C, PTPRD, CALR, S100A7, CDH5, CACNA2D1, PTPRS, NEGR1 and SMPDL3A or a gene encoding the same is decreased compared to a control group.

[0076] 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.

[0077] 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.

[0078] In one specific example, the method for providing information for predicting or diagnosing gestational diabetes can be performed using a plurality of panels.

[0079] A composition for predicting or diagnosing gestational diabetes according to a certain aspect or a method for providing information for predicting or diagnosing the same can simply and effectively predict or diagnose gestational diabetes 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.

[0080] Figure 1 shows the results of a heat map analysis of the levels of early pregnancy gestational diabetes-specific biomarkers selected for provisional early pregnancy gestational diabetes prediction or diagnosis.

[0081] Figure 2 shows the results of a heat map analysis of the levels of mid-trimester gestational diabetes-specific biomarkers selected for the prediction or diagnosis of interim mid-trimester gestational diabetes.

[0082] Figure 3 shows the results of a heat map analysis of the levels of late-term gestational diabetes-specific biomarkers selected for provisional diagnosis of late-term gestational diabetes.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] Throughout the specification of the present invention, “A and / or B” means A or B, or A and B.

[0087]

[0088] Example 1. Extraction of plasma proteins from early, mid, and late pregnancy maternal blood.

[0089] Plasma proteins were extracted from maternal plasma samples from 10 normal pregnant women and 10 pregnant women with gestational diabetes in the first, second, and third trimesters of pregnancy.

[0090]

[0091] 1.1 Plasma depletion

[0092] 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.

[0093]

[0094] 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-

[0095]

[0096] 1.2 Protein lysis and digestion

[0097] Protein dissolution and digestion are performed in the following steps (1) to (4).

[0098] (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.

[0099] (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.

[0100] (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.

[0101] (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.

[0102]

[0103] Example 2. Selection of biomarkers for predicting or diagnosing gestational diabetes in early pregnancy.

[0104] To select a biomarker for predicting or diagnosing gestational diabetes 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.

[0105] 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.

[0106]

[0107] LC & LC-MS / MS systemNano-LC Ultimate 3000 & Thermo Orbitrap Exploris 480ColumnTrap column: Acclaim PepMap TM 100, 100 ㎛ x 2 ㎝Analytical column: PepMap TMRSLC 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 %

[0108]

[0109] 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.

[0110]

[0111] Search parameters are as follows:

[0112] - 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)

[0113] - MBR (match-between-runs) mode.

[0114]

[0115] 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 applied 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 gestational diabetes), protein signatures satisfying the following criteria (1) to (5) were defined:

[0116] When placed into two groups for comparison,

[0117] (1) Retain only proteins with missing value <=50% in each group.

[0118] (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.

[0119] (3) empirical p-value <0.05,

[0120] (4) median expression in the condition of interest >0, and

[0121] (5) median expression in the other conditions <0.

[0122]

[0123] As a result of the performance, proteins showing final expression changes were selected as biomarkers for predicting or diagnosing gestational diabetes in early pregnancy.

[0124]

[0125] Figure 1 shows the results of a heat map analysis of the levels of early pregnancy gestational diabetes-specific biomarkers selected for provisional early pregnancy gestational diabetes prediction or diagnosis.

[0126] As shown in Figure 1, GSK3A, FCGR3B, C3, LDLR, FN1, P4HB, TUBB, COL1A2, SELL, GPT, SERPINB3, DDT, ERAP2, PRAP1, PAPPA2, and DPP3 showed relatively higher levels in the early pregnancy gestational diabetes group compared to the control group.

[0127] For FBLN1, SDCBP, TREH, SERPINE2, UMOD, SLC3A2, HLA-C, LIPC, PTPRD, RAN, CTBS, GALNT1, DSG2, NECTIN1, and GDF15, relatively lower levels were observed in the early pregnancy gestational diabetes group compared to the control group.

[0128]

[0129] Example 3. Selection of biomarkers for predicting or diagnosing mid-trimester gestational diabetes.

[0130] To select a biomarker for predicting or diagnosing mid-pregnancy gestational diabetes, analysis was performed using mass spectrometry-based LC / MS after protein extraction from mid-pregnancy maternal plasma according to Example 1.

[0131] 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-pregnancy gestational diabetes.

[0132]

[0133] Figure 2 shows the results of a heat map analysis of the levels of mid-trimester gestational diabetes-specific biomarkers selected for the prediction or diagnosis of interim mid-trimester gestational diabetes.

[0134] As shown in Figure 2, for IGHA1, CRP, LTF, GPI, RET, COL1A2, GSTP1, MMP9, TYMP, PTPRB, RNASE4, CAMP, PPIA, ENPEP, DSC3, ERAP2, MEGF8, OIT3 and TSEN34, the levels were relatively higher in the mid-trimester gestational diabetes group compared to the control group.

[0135] For CHGA, CR2, CAST, GFUS, FLNC, VASN, FERMT3, KCTD12, PRAP1, PGLYRP2, ERVMER34-1, SLC38A10, MYRF, and RFNG, relatively lower levels were observed in the mid-trimester gestational diabetes group compared to the control group.

[0136]

[0137] Example 4. Selection of biomarkers for diagnosing gestational diabetes in late pregnancy.

[0138] To select a biomarker for diagnosing gestational diabetes in late pregnancy, protein extraction from maternal plasma in late pregnancy according to Example 1 was performed followed by analysis using mass spectrometry-based LC / MS.

[0139] 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 diagnosing gestational diabetes in late pregnancy.

[0140]

[0141] Figure 3 shows the results of a heat map analysis of the levels of late-term gestational diabetes-specific biomarkers selected for provisional diagnosis of late-term gestational diabetes.

[0142] As shown in Figure 3, for SVOPL, APOM, KLKB1, SOD2, PSG1, COL6A2, TCN1, PTPRM, CHI3L1, B2M and LTBP1, the levels were relatively higher in the late pregnancy gestational diabetes group compared to the control group.

[0143] For NECTIN2, RNASET2, ISLR, CUTA, LILRB5, SOD1, HLA-C, PTPRD, CALR, S100A7, CDH5, CACNA2D1, PTPRS, NEGR1, and SMPDL3A, relatively lower levels were observed in the late-term gestational diabetes group compared to the control group.

[0144]

[0145] 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.

[0146]

[0147] The present invention relates to a disease-specific biomarker for early prediction and diagnosis of gestational diabetes and its use, and can be used for early prediction and diagnosis of gestational diabetes.

[0148] Related national research and development projects

[0149] - Assignment ID: 1465039723

[0150] - Assignment Number: RS-2022-KH129953

[0151] - Ministry name: Ministry of Health and Welfare

[0152] - Project Management (Professional) Organization Name: Korea Health Industry Development Institute

[0153] - Research Project Name: Research-Oriented Hospital Development

[0154] Research Project Name: Establishing a Platform to Address Unmet Pregnancy and Childbirth Medical Needs Based on Future Innovation Technology

[0155] - Project implementation organization name: Bundang Cha Hospital

[0156] - Research period for the current year: January 1, 2023 - December 31, 2023

Claims

1. A biomarker composition for predicting or diagnosing mid-trimester gestational diabetes, comprising at least one protein selected from the group consisting of IGHA1, CRP, LTF, GPI, RET, COL1A2, GSTP1, MMP9, TYMP, PTPRB, RNASE4, CAMP, PPIA, ENPEP, DSC3, ERAP2, MEGF8, OIT3, TSEN34, CHGA, CR2, CAST, GFUS, FLNC, VASN, FERMT3, KCTD12, PRAP1, PGLYRP2, ERVMER34-1, SLC38A10, MYRF and RFNG or a gene encoding the same.

2. A composition for predicting or diagnosing mid-trimester gestational diabetes, comprising an agent for measuring the level of one or more proteins selected from the group consisting of IGHA1, CRP, LTF, GPI, RET, COL1A2, GSTP1, MMP9, TYMP, PTPRB, RNASE4, CAMP, PPIA, ENPEP, DSC3, ERAP2, MEGF8, OIT3, TSEN34, CHGA, CR2, CAST, GFUS, FLNC, VASN, FERMT3, KCTD12, PRAP1, PGLYRP2, ERVMER34-1, SLC38A10, MYRF and mRNA of a gene encoding the same.

3. A composition for predicting or diagnosing mid-trimester gestational diabetes, 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 gestational diabetes, 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 PNA (peptide nucleic acid) and an antisense nucleotide that specifically bind to the gene.

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

6. A kit for predicting or diagnosing mid-trimester gestational diabetes, wherein the kit according to claim 5 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 IGHA1, CRP, LTF, GPI, RET, COL1A2, GSTP1, MMP9, TYMP, PTPRB, RNASE4, CAMP, PPIA, ENPEP, DSC3, ERAP2, MEGF8, OIT3, TSEN34, CHGA, CR2, CAST, GFUS, FLNC, VASN, FERMT3, KCTD12, PRAP1, PGLYRP2, ERVMER34-1, SLC38A10, MYRF and a gene encoding the same 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 gestational diabetes mellitus in 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 gestational diabetes, 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, orchite 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 gestational diabetes in 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 IGHA1, CRP, LTF, GPI, RET, COL1A2, GSTP1, MMP9, TYMP, PTPRB, RNASE4, CAMP, PPIA, ENPEP, DSC3, ERAP2, MEGF8, OIT3 and TSEN34 or a gene encoding the same is increased compared to the control group, it is determined to be gestational diabetes, or A method for determining gestational diabetes further comprises a step of determining that the level of mRNA of one or more proteins selected from the group consisting of CHGA, CR2, CAST, GFUS, FLNC, VASN, FERMT3, KCTD12, PRAP1, PGLYRP2, ERVMER34-1, SLC38A10, MYRF and RFNG or a gene encoding the same is decreased compared to the control group. A method for providing information for predicting or diagnosing gestational diabetes mellitus in the second trimester of pregnancy.

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