Composition for diagnosis of lung damage caused by humidifier disinfectant or exposure to humidifier disinfectant components and use thereof

Abstract

According to an invention relating to a diagnostic composition capable of predicting exposure to humidifier disinfectant components and the use thereof, and according to one aspect, a diagnostic composition capable of predicting lung damage caused by a humidifier disinfectant or exposure to humidifier disinfectant components and a method for providing information using the same, proteins or genes showing differences in expression levels between a normal group not exposed to humidifier disinfectants and a group with lung disease caused by humidifier disinfectants, or between a normal group not exposed to humidifier disinfectants and an exposed group, can be identified, and lung damage caused by humidifier disinfectants or exposure to humidifier disinfectant components can be predicted through analysis of an XGBoost prediction model using the identified proteins or genes.

Description

Technology Field

[0001] This invention relates to a composition for diagnosing lung damage caused by humidifier disinfectants or exposure to humidifier disinfectant components, and the use thereof.

[0002] This patent was carried out with the support of the National Institute of Environmental Research, funded by the Ministry of Environment (NIER-2019-04-02-077). Background Technology

[0003] PGH (oligo-[2-(2-ethoxy)-ethoxyeth-yl)-guanidinium-chloride), PHMG (polyhexamethylene guanidine), and PHMB (polyhexamethylene biguanide) have been widely used as anti-infective and antimicrobial agents due to their known excellent bactericidal activity. These components exhibit a bactericidal effect by destroying the phospholipid bilayer, but it has been confirmed that they may destroy the phospholipids of human cells, pose a risk of damaging the skin barrier, and have inhalation toxicity.

[0004] However, as the above ingredients were used in humidifier disinfectants, victims suffered lung damage caused by the disinfectant ingredients, and research is being conducted to determine the causal relationship between humidifier disinfectants and the affected diseases. Specifically, epidemiological, toxicological, and clinical studies are being conducted to identify diseases caused by humidifier disinfectants.

[0005] Epidemiological research is a discipline that logically investigates the factors involved in bringing about results by classifying them according to human characteristics such as gender, age, occupation, and nutritional status, as well as geographical location and time. Through epidemiological research, it is possible to identify the distribution patterns of diseases within human populations and the causes that determine these distribution patterns. However, in the case of the humidifier disinfectant incident, there were instances where cases of clinical health damage were observed even though there was no clear statistically significant association between exposure to humidifier disinfectants and the affected diseases (Park & ​​Ku, 2020). Furthermore, from a toxicological perspective, the actual usage of humidifier disinfectants is difficult to reconstruct because individual exposure varies as households do not use them according to a consistent dosage regimen (Park et al., 2015; Park, 2018), and there are limitations in responding to the identification of the continuously expanding affected diseases. Moreover, as time passes from the time of exposure to humidifier disinfectants, traces in lung imaging that are recognized as evidence for directly identifying causality disappear, and there is a problem in that it is difficult to accurately assess damage due to the recovery of lung function.

[0006] Therefore, there is an urgent need to provide a biomarker capable of diagnosing lung damage caused by humidifier disinfectants or exposure to humidifier disinfectant components, a composition containing the same, and a method for providing information on lung damage caused by humidifier disinfectants or exposure to humidifier disinfectant components using the same. The problem to be solved

[0007] One aspect provides a diagnostic composition capable of predicting lung damage caused by humidifier disinfectants or predicting exposure to humidifier disinfectant components, comprising a preparation capable of measuring the expression level of one or more proteins or genes selected from the group consisting of F13B, ITIH4, POTEF, APOM, APOL1, HPR, APOF, FN1, SAA4, and APOB.

[0008] Another aspect is to provide a kit for predicting lung damage caused by a humidifier disinfectant containing the above composition or for predicting exposure to humidifier disinfectant components.

[0009] Another aspect provides a method for providing information for predicting lung damage caused by a humidifier disinfectant or predicting exposure to a humidifier disinfectant component, comprising: a step of measuring the expression level of one or more proteins or genes selected from a group consisting of F13B, ITIH4, POTEF, APOM, APOL1, HPR, APOF, FN1, SAA4, and APOB in a biological sample isolated from a target individual; and a step of generating a score by inputting a specific weight into an algorithm based on the expression level of said protein or gene, and diagnosing that there is a high probability of lung damage caused by a humidifier disinfectant or exposure to a humidifier disinfectant component if the score is higher than that of a control group not exposed to the humidifier disinfectant. means of solving the problem

[0010] One aspect provides a diagnostic composition capable of predicting lung damage caused by humidifier disinfectants or predicting exposure to humidifier disinfectant components, comprising a preparation capable of measuring the expression level of one or more proteins or genes selected from the group consisting of F13B, ITIH4, POTEF, APOM, APOL1, HPR, APOF, FN1, SAA4, and APOB.

[0011] The term "humidifier disinfectant" in this specification refers to a guanine-based compound used to prevent the proliferation of microorganisms in a humidifier. Specifically, the humidifier disinfectant is included in the spray liquid of a humidifier, and the humidifier disinfectant component may be selected from the group consisting of PHMG (polyhexamethylene guanidine), PGH (oligo-[2-(2-ethoxy)-ethoxyeth-yl)-guanidinium-chlorid), and PHMB (polyhexamethylene biguanide), but is not limited thereto. Specifically, among the components of humidifier disinfectants, PHMG-p (Polyhexamethylene guanidine-phosphate) and PGH (Oligo(2-(2-ethoxy)ethoxyethyl guanidine chloride) are substances with similar structures belonging to the guanidine family. They are broad-spectrum antimicrobial agents frequently used in various consumer products, such as shampoos, skin preservatives, and disinfectants for water systems and swimming pools, due to their reported safety regarding oral or skin routes; however, it has been confirmed that inhaling them causes diseases associated with lung damage, such as interstitial lung disease, pulmonary fibrosis, and asthma (Park et al., 2016; Leem & Lee, 2017).

[0012] The term "lung damage caused by humidifier disinfectants" in this specification refers to lung damage that occurs when the guanine-based compounds are inhaled. The guanine-based compounds may include, but are not limited to, PHMG (polyhexamethylene guanidine), PGH (oligo-[2-(2-ethoxy)-ethoxyeth-yl)-guanidinium-chlorid), PHMB (polyhexamethylene biguanide), etc. Specifically, lung damage that occurs when the guanine-based compounds are inhaled may include, but is not limited to, interstitial lung disease, pulmonary fibrosis, asthma, bronchiectasis, pneumonia, etc.

[0013] The term "diagnosis" in this specification may include determining the susceptibility of a subject to a specific disease or condition, determining whether the subject currently has a specific disease or condition, determining the prognosis of a subject with a specific disease or condition, or therametrics (e.g., monitoring the condition of an object to provide information on therapeutic efficacy). Specifically, the diagnosis is to predict lung damage caused by humidifier disinfectants or to predict whether there has been exposure to humidifier disinfectant components. The prediction refers to a prospect or preliminary evaluation regarding the progression of the disease or recovery. Specifically, the prediction is to determine whether there has been exposure to humidifier disinfectant components or lung damage caused by humidifier disinfectants in subjects at risk of exposure to humidifier disinfectants.

[0014] In this specification, the term "biomarker" or "marker" refers to a substance capable of distinguishing between a normal group not exposed to humidifier disinfectants and a group that has developed lung disease caused by humidifier disinfectants, or between a normal group not exposed to humidifier disinfectants and an exposed group; it may include all organic biomolecules such as polypeptides, proteins, nucleic acids, genes, lipids, glycolipids, glycoproteins, or sugars that show an increase or decrease in individuals at risk of exposure to humidifier disinfectants. In one embodiment, it may be a protein whose expression level changes in individuals at risk of exposure to humidifier disinfectants, but is not limited thereto.

[0015] The terms in this specification, “F13B (Coagulation factor XIII B chain), APOF (Apolipoprotein F), ITIH4 (Inter-alpha-trypsin inhibitor heavy chain H4), FN1 (Fibronectin), POTEF (POTE ankyrin domain family member F), APOM (Apolipoprotein M), APOL1 (Apolipoprotein L1), SAA4 (Serum amyloid A-4 protein), HPR (Haptoglobin-related protein) and APOB (Apolipoprotein B-100)” are genes or proteins that show differences in expression in blood analysis between a normal group not exposed to humidifier disinfectants and a group with lung disease caused by humidifier disinfectants, or between a normal group not exposed to humidifier disinfectants and a group exposed to humidifier disinfectants.

[0016] In this specification, the term "measurement of gene expression level" refers to a process of confirming the degree of mRNA expression of a marker gene capable of predicting lung damage caused by humidifier disinfectants or whether there has been exposure to humidifier disinfectant components, and can be performed by measuring the amount of mRNA. Analysis methods for measuring the mRNA level include, but are not limited to, 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), RNase protection assay, Northern blotting, or DNA microarray chip (DNA chip).

[0017] The term "preparation for measuring the expression level of a gene" in this specification refers to a molecule that can be used to detect a marker by confirming the expression level of the mRNA or protein of a marker whose expression level increases or decreases depending on lung damage caused by the humidifier disinfectant or exposure to the humidifier disinfectant component.

[0018] In one embodiment, the agent capable of measuring the gene expression level may comprise one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to one or more genes selected from the group consisting of F13B, ITIH4, POTEF, APOM, APOL1, and HPR, but is not limited thereto. Preferably, the agent capable of measuring the gene expression level may further comprise one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to one or more genes selected from the group consisting of APOF, FN1, SAA4, and APOB, but is not limited thereto.

[0019] The term “primer” in this specification refers to any combination of primer pairs consisting of forward and reverse primers that recognize a target gene sequence, and preferably refers to a primer pair that provides analytical results having specificity and sensitivity. The primer refers to a single-stranded oligonucleotide capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (i.e., the presence of four different nucleoside triphosphates, polymerase, temperature, buffer, etc.). The suitable length of the primer varies depending on various factors, such as temperature and the application of the primer, but is typically 15 to 30 nucleotides.

[0020] The sequence of the above primer does not need to be completely complementary to a portion of the template sequence; it is sufficient to have sufficient complementarity within the range where it can hybridize with the template and perform its inherent function. Additionally, the above primer can be prepared with a sequence complementary to the mRNA (i.e., cDNA) sequence of the target gene. The design of such a primer can be easily carried out by a person skilled in the art by referring to the cDNA sequence of the target gene.

[0021] The term "probe" in this specification refers to a substance capable of specifically binding to a target substance to be detected within a biological sample, and means a substance capable of specifically confirming the presence of the target substance within the sample through said binding. Specifically, the probe is a substance commonly used in the art and can be produced in the form of an oligonucleotide probe, a single-strand DNA probe, a double-strand DNA probe, an RNA probe, etc. Specifically, it may be a nucleic acid fragment such as RNA or DNA corresponding to a few bases or as long as several hundred bases, and is labeled so as to confirm the presence or absence and expression level of a specific mRNA.

[0022] The above primer or probe can be chemically synthesized using a phosphoramidite solid support synthesis method or other widely known methods. In addition, the above primer or probe can be modified through various methods known in the art. Examples of such modifications include methylation, capping, substitution with one or more homologues of natural nucleotides, and modification between nucleotides, for example, modification to uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.).

[0023] The term "antisense oligonucleotide" in this specification refers to DNA or RNA or derivatives thereof comprising a nucleic acid sequence complementary to a specific mRNA sequence, which acts to inhibit the translation of the mRNA into a protein by binding to the complementary sequence within the mRNA. An antisense oligonucleotide sequence refers to a DNA or RNA sequence that is complementary to the mRNA of the said genes and capable of binding to said mRNA. This can inhibit essential activities for the translation, translocation into the cytoplasm, maturation, or any other overall biological function of said gene mRNA. The length of the antisense oligonucleotide may be 6 to 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases. The antisense oligonucleotide may be synthesized in vitro by conventional methods and administered in vivo, or the antisense oligonucleotide may be synthesized in vivo.

[0024] The term "preparation for measuring the expression level of a protein" in this specification refers to a molecule that can be used to detect a marker by confirming the expression level of a protein, which is a marker whose expression level increases or decreases depending on lung damage caused by the humidifier disinfectant or exposure to the humidifier disinfectant component.

[0025] In one embodiment, the agent for measuring the activity level of the protein may comprise one or more selected from the group consisting of an antibody that specifically recognizes one or more proteins selected from the group consisting of F13B, ITIH4, POTEF, APOM, APOL1, and HPR, an oligopeptide, a ligand, a PNA (peptide nucleic acid), and an aptamer, but is not limited thereto. Preferably, the agent for measuring the activity level of the protein may comprise one or more selected from the group consisting of an antibody that specifically recognizes one or more proteins selected from the group consisting of APOF, FN1, SAA4, and APOB, an oligopeptide, a ligand, a PNA (peptide nucleic acid), and an aptamer, but is not limited thereto.

[0026] The term "antibody" in this specification refers to a substance that specifically binds to an antigen to induce an antigen-antibody reaction. The antibody may comprise not only a complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of the antibody molecule. A functional fragment of the antibody molecule refers to a fragment possessing at least an antigen-binding function and may be, for example, Fab, F(ab'), F(ab')2, or Fv. Furthermore, the antibody includes polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Additionally, the antibody may be easily manufactured using techniques widely known in the art. The polyclonal antibody may be produced from any animal, such as a goat, rabbit, sheep, monkey, horse, pig, cattle, or dog. The above monoclonal antibody can be prepared using the hybridoma method (see Kohler and Milstein (1976) European Journal of Immunology 6:511-519), which is widely known in the art, or phage antibody library technology (see Clackson et al, Nature, 352:624-628, 1991; Marks et al, J. Mol. Biol., 222:58,1-597, 1991).

[0027] The term "PNA (Peptide Nucleic Acid)" in this specification refers to an artificially synthesized polymer similar to DNA or RNA. Specifically, unlike DNA, which has a phosphate-ribose sugar backbone, PNA has a repeating N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which can significantly increase its binding strength and stability to DNA or RNA.

[0028] The term "aptamer" in this specification means a nucleic acid (DNA, RNA) or peptide that can affinityally and specifically bind to a target molecule.

[0030] Another aspect provides a kit for predicting lung damage caused by a humidifier disinfectant or for predicting exposure to a humidifier disinfectant component, comprising the above composition.

[0031] The above-mentioned humidifier disinfectant, lung damage caused by the humidifier disinfectant, and preparations for measuring the level of gene or protein expression are as described above.

[0032] The above kit may be an RT-PCR (Reverse transcription polymerase chain reaction) kit, a DNA chip kit, an ELISA (Enzymelinked immunosorbent assay) kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit, but is not limited thereto. Additionally, the above kit may be configured to include one or more other components, solutions, or devices suitable for the analysis method.

[0033] For example, the RT-PCR kit may include a pair of primers specific to the marker gene, and may also include a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC-water, sterile water, etc. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or its fragment are attached, and reagents, preparations, enzymes, etc., for producing a fluorescent probe, and the substrate may include cDNA or oligonucleotides corresponding to a control gene or its fragment. The ELISA kit includes an antibody specific to the protein. The antibody is an antibody with high specificity and affinity for the marker protein and minimal cross-reactivity with other proteins, and is a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. Other ELISA kits may include reagents capable of detecting conjugated antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with antibodies), their substrates, or other substances capable of binding to antibodies. The protein chip kit may be a kit in which one or more antibodies against a marker are arranged at a fixed location on a substrate and immobilized at a high density. The protein chip may be used to isolate proteins from a biological sample, hybridize the isolated proteins with the protein chip to form an antigen-antibody complex, and read the complex to confirm the presence or expression level of the protein. In this specification, the term "antigen-antibody complex" refers to a combination of a corresponding protein antigen in a biological sample and an antibody that recognizes it.The above antigen-antibody complex may be detected using methods known in the art, such as spectroscopic, photochemical, biochemical, immunochemical, electrochemical, spectrophotometric, chemical, and other methods. The rapid kit may include an antibody specific to the protein and / or an antibody specific to a control protein. Additionally, the rapid kit may include other materials capable of detecting the bound antibody, such as a reagent, for example, a nitrocellulose membrane with the specific antibody and secondary antibody immobilized thereon, a membrane bound to an antibody-bound bead, an absorbent pad, and a sample pad.

[0034] In one embodiment, the kit may be an integrated magneto-electrochemical sensor kit, but is not limited thereto. 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 attaches a substance capable of detecting a target substance (e.g., an antibody) to magnetic beads and detects an electrical signal by enzymatic signal amplification generated by a chromogenic substance capable of detecting the target substance (e.g., horseradish peroxidase (HRP), alkaline phosphatase (ALP), α-D-galactosidase (α-Gal).

[0035] In one embodiment, the kit may include a computer embedded with a formulation, a device, and an algorithm for predicting lung damage caused by the humidifier disinfectant or whether there is exposure to a humidifier disinfectant component, and may relate to a kit that associates the measurement results of the marker level with allergic rhinitis through the algorithm. More specifically, the kit may include a diagnostic composition capable of predicting lung damage caused by the humidifier disinfectant or whether there is exposure to a humidifier disinfectant component, comprising a formulation capable of measuring the expression level of one or more proteins or genes selected from the group consisting of F13B, ITIH4, POTEF, APOM, APOL1, and HPR, and preferably may include a diagnostic composition capable of predicting lung damage caused by the humidifier disinfectant or whether there is exposure to a humidifier disinfectant component, further comprising a formulation capable of measuring the expression level of one or more proteins or genes selected from the group consisting of APOF, FN1, SAA4, and APOB, but is not limited thereto.

[0037] Another aspect provides a method for providing information for predicting lung damage caused by a humidifier disinfectant or predicting exposure to a humidifier disinfectant component, comprising: a step of measuring the expression level of one or more proteins or genes selected from a group consisting of F13B, ITIH4, POTEF, APOM, APOL1, HPR, APOF, FN1, SAA4, and APOB in a biological sample isolated from a target individual; and a step of generating a score by inputting a specific weight into an algorithm based on the expression level of said protein or gene, and diagnosing that there is a high probability of lung damage caused by a humidifier disinfectant or exposure to a humidifier disinfectant component if the score is higher than that of a control group not exposed to the humidifier disinfectant.

[0038] The above-mentioned humidifier disinfectant, lung damage caused by the humidifier disinfectant, and preparations for measuring the level of gene or protein expression are as described above.

[0039] The term "intended subject" in this specification refers to a subject at risk of exposure to humidifier disinfectants, meaning a subject for which it is uncertain whether there is lung damage caused by the humidifier disinfectant or whether there has been exposure to the components of the humidifier disinfectant.

[0040] In this specification, the term "biological sample" may be a sample isolated from an individual at risk of exposure to humidifier disinfectants, cultured cells, or feces. Specifically, the biological samples include bronchial biopsy, lung biopsy, nasal cavity biopsy, sinus biopsy, sputum, bronchoalveolar lavage fluid (BALF), nasal lavage fluid, nasal swap, nasal aspirate, nasopharyngeal swab, nasopharyngeal aspirate, nasopharyngeal lavage fluid, whole blood, plasma, serum, tears, mucus, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, and lymph. It may be fluid), pleural fluid, nipple aspirate, synovial fluid, joint aspirate, organ secretions, cell, cell extract, cerebrospinal fluid, or a combination thereof, and preferably may be selected from the group consisting of blood, serum and plasma, but is not limited thereto.

[0041] Methods for measuring the expression level of the above protein include, but are not limited to, western blotting, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, fluorescence-activated cell sorter analysis (FACS), protein chip technology assay, or DDA (Data dependent acquisition) using LC-MS or SWATH (Sequential Window Acquisition of all Theoretical Mass Spectra).

[0042] Methods for measuring the expression level of the above gene include, but are not limited to, 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), RNase protection assay, Northern blotting, or DNA microarray chip (DNA chip).

[0043] In this specification, the term "increase in the level of protein expression" means that the expression of protein in the sample of the target individual is significantly increased to a measurable level compared to a normal group not exposed to humidifier disinfectants, and may mean, for example, an increase of 1.1 times or more. For example, it may mean an increase of 1.1 to 2.5 times, specifically, 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, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, or 2.5 times.

[0044] In this specification, the term “increase in the level of gene expression” means that the concentration of mRNA in the sample of the target individual is significantly increased to a measurable level compared to a normal group not exposed to humidifier disinfectants, and may mean, for example, an increase of 1.1 times or more. For example, it may mean an increase of 1.1 to 2.5 times, specifically, 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, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, or 2.5 times.

[0045] In this specification, the term “reduction in the level of protein activity” means that the activity of a protein in a sample of an individual is significantly reduced to a measurable level compared to a normal group not exposed to humidifier disinfectants, and may mean, for example, a reduction of 0.1 times or less. For example, it may mean a reduction of 0.1 times to 0.9 times, specifically, a reduction of 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, or 0.9 times.

[0046] In this specification, the term “reduction in the level of gene expression” means that the concentration of mRNA in a sample of an individual is significantly reduced to a measurable level compared to a normal group not exposed to humidifier disinfectants, and may mean, for example, a reduction of 0.1 times or less. For example, it may mean a reduction of 0.1 times to 0.9 times, specifically, a reduction of 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, or 0.9 times.

[0047] In one embodiment, the method for providing information for predicting lung damage caused by the humidifier disinfectant or for predicting exposure to the humidifier disinfectant component may further include, but is not limited to, the step of producing a score by calculating a specific weight on the expression level of one or more proteins or genes selected from the group consisting of the measured F13B, ITIH4, POTEF, APOM, APOL1, HPR, APOF, FN1, SAA4, and APOB; and the step of comparing the score with a threshold value.

[0048] The term "weight" in this specification refers to the importance assigned to individual values ​​when calculating an average value, and a unique specific weight may be assigned to each protein or gene that shows a difference in expression between the normal group not exposed to humidifier disinfectant and the group with lung disease caused by humidifier disinfectant, or between the normal group not exposed to humidifier disinfectant and the exposed group. Specifically, the weights of the proteins F13B, ITIH4, POTEF, APOM, APOL1, HPR, APOF, FN1, SAA4, or APOB may be F13B (3.08), ITIH4 (1.96), POTEF (-1.06), APOM (-2.40), APOL1 (-2.60), HPR (-3.72), APOF (3.04), FN1 (1.04), SAA4 (-2.99), or APOB (-7.48).

[0049] The term "score" in this specification may refer to a value calculated by assigning specific weights to the expression level of a protein or gene. The score may be calculated according to a machine learning process.

[0050] The term "threshold" in this specification may refer to a value on the line corresponding to the significance level α value in the distribution of the test statistic. Specifically, in the method for providing information for predicting lung damage caused by humidifier disinfectants, the threshold value may be 25.551 to 36.299, and in the method for providing information for predicting exposure to humidifier disinfectant components, the threshold value may be 28.192 to 36.977, but is not limited thereto.

[0051] The step of producing the above score; and the step of comparing the above score with a threshold value; may utilize an algorithm. The algorithm may be at least one model among XGBoost (eXtreme Gradient Boosting), MLP (multi-layer perceptron), SVM (support vector machine), RF (random forest), an ensemble model composed of a combination of multiple models, and a pre-trained model, but is not limited thereto.

[0052] In one embodiment, the method for providing information for predicting lung damage caused by the humidifier disinfectant or for predicting exposure to the humidifier disinfectant component may further include the step of determining that the subject has lung damage caused by the humidifier disinfectant or has been exposed to the humidifier disinfectant component when the score is greater than or equal to the threshold value; but is not limited thereto.

[0053] In one embodiment, the method may additionally use clinical information other than the marker of an individual in addition to the results of measuring and analyzing the level of the gene to provide information for predicting lung damage caused by a humidifier disinfectant or for predicting exposure to a humidifier disinfectant component. Such clinical information includes, for example, the patient's age, gender, weight, dietary habits, body mass, ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), angiography, etc. Effects of the invention

[0054] According to a diagnostic composition capable of predicting lung damage caused by humidifier disinfectants or exposure to humidifier disinfectant components based on a pattern, and a method for providing information using the same, it is possible to identify proteins or genes such as F13B, ITIH4, POTEF, APOM, APOL1, HPR, APOF, FN1, SAA4, or APOB that show differences in expression between a normal group not exposed to humidifier disinfectants and a group with lung disease caused by humidifier disinfectants, or between a normal group not exposed to humidifier disinfectants and an exposed group, and to predict lung damage caused by humidifier disinfectants or exposure to humidifier disinfectant components through XGBoost model analysis using the same. Brief explanation of the drawing

[0055] Figure 1 is a graph showing a model that can distinguish between a normal group not exposed to humidifier disinfectants and a group with lung disease caused by humidifier disinfectants (stages 1, 2, 3, and 4) using an XGBoost analysis model containing 10 protein patterns in children. Figure 2 is a graph showing the results of verifying whether the normal group not exposed to humidifier disinfectants and the exposed group can be distinguished using an XGBoost analysis model containing 10 protein patterns in children. Figure 3 is a graph showing the results of verifying whether a normal group not exposed to humidifier disinfectants and clinically almost certain stage 1 lung disease caused by humidifier disinfectants can be distinguished using an XGBoost analysis model containing 10 protein patterns in children. Figure 4 is a graph showing the results of verifying whether the normal group not exposed to humidifier disinfectants and the humidifier disinfectant lung disease group (stages 1, 2, 3, and 4) can be distinguished using an XGBoost analysis model containing 10 protein patterns in adults. Figure 5 is a graph showing the results of verifying whether the normal group not exposed to humidifier disinfectants and the exposed group can be distinguished using an XGBoost analysis model containing 10 protein patterns in adults. Figure 6 is a graph showing the results of verifying whether a normal group not exposed to humidifier disinfectants and a patient group with clinically almost certain stage 1 lung disease caused by humidifier disinfectants can be distinguished using an XGBoost analysis model containing 10 protein patterns in adults. Specific details for implementing the invention

[0056] The following examples will be explained in more detail. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0057] Example 1. Characteristics of the subject and preparation of the blood sample

[0058] To determine exposure to humidifier disinfectant components and to select biomarkers for classifying the stage of lung damage caused by exposure, a patient group exposed to humidifier disinfectants and a non-exposed normal group not exposed to humidifier disinfectants were recruited, and their plasma proteins were analyzed.

[0059] The demographic and clinical characteristics of the subjects participating in the experiment are as follows.

[0060] Specifically, the experiment was conducted by dividing participants into children and adults. The characteristics of the pediatric subjects are shown in Table 1 below.

[0061] Non-exposed normal group Step 1 Step 2 Step 3 Step 4 Number of subjects 162 42 54 51 76 Average age 7.88 8.83 10.44 9.08 11.14 Gender (Male / Female) 87 / 75 26 / 16 24 / 30 26 / 25 49 / 27

[0062] The characteristics of the adult subjects are as shown in Table 2 below.

[0063] Non-exposed normal group Step 1 Step 2 Step 3 Step 4 Number of subjects 50 14 6 7 20 Average age 33.68 39.50 39.33 39.29 36.55 Gender (Male / Female) 0 / 50 0 / 14 0 / 6 0 / 7 0 / 20

[0064] Example 2. Classification of lung damage stages caused by exposure to humidifier disinfectant components

[0065] The severity of damage to patients exposed to humidifier disinfectants was classified into stages 1 to 4 based on the assessment. Specifically, stage 1 was classified as cases where the cause of the disease was almost certainly humidifier disinfectant, stage 2 as cases where the cause of the disease was highly likely to be humidifier disinfectant, stage 3 as cases where the cause of the disease was low likely to be humidifier disinfectant, and stage 4 as cases where the cause of the disease was almost impossible to be humidifier disinfectant.

[0066] The plasma samples from stages 1 to 4 above were provided by the Humidifier Disinfectant Health Center at Asan Medical Center, and the plasma samples used were from subjects who were exposed to PGH (oligo-[2-(2-ethoxy)-ethoxyeth-yl)-guanidinium chloride) or PHMG (polyhexamethylene guanidine) by more than 50% among the humidifier disinfectant components.

[0067] Example 3. Quantitative plasma proteomic analysis and derivation of biomarkers associated with humidifier disinfectant-induced lung injury or humidifier disinfectant exposure

[0068] 3.1 Quantitative Plasma Proteomic Analysis

[0069] In order to identify biomarkers associated with lung damage or exposure to humidifier disinfectants from selected plasma samples, plasma proteins were quantitatively analyzed.

[0070] Specifically, SWATH (Sequential Window Acquisition of all Theoretical Mass Spectra), a quantitative plasma proteomics analysis technique based on Quadrupole Time of Flight (QF) mass spectrometry, was used to quantitatively analyze plasma proteins from a total of 482 plasma samples. Using mixed samples, DPHL: A pan-human protein mass spectrometry library (Reference paper: https: / / pubmed.ncbi.nlm.nih.gov / 32795611 / Using the spectral library, a small amount of 40 mL of blood sample was pretreated with cleaved peptides, and plasma proteins were quantitatively analyzed through LC / MS SWATH analysis.

[0071] Before applying 482 plasma samples to SWATH analysis, a pretreatment process was performed to ensure reproducible analysis. This involved separating low-concentration proteins using a MARS14 column (100 x 4.6 mm; Agilent Technology, Palo Alto, CA, USA) connected to High-performance liquid chromatography (HPLC) and preparing peptides using an automated liquid dispensing system (Bravo Automated Liquid Handling Platform - Agilent). Based on the quantification values ​​obtained using NanoDrop after peptide preparation, 482 different samples were analyzed in equal amounts using SWATH-MS.

[0072] More specifically, 40 mL of plasma was injected into a MARS14 column to remove the top 14 abundant proteins (albumin, IgA, IgG, IgM, α1-antitrypsin, α1-acid glycoprotein, apolipoprotein A1, apolipoprotein A2, complement C3, transferrin, α2-macroglobulin, transthyretin, haptoglobin, and fibrinogen). For this purpose, the mixture was diluted 4-fold with Agilent buffer A (part number: 5185-5987) and loaded onto a MARS14 column of a Shimadzu HPLC LC20AT system. The fraction of unbound, low-concentration plasma proteins was used to perform automated filter-aided sample preparation (FASP) using an automated liquid dispensing system. A vacuum was supplied during sample preparation using a Multiscreen Vacuum Manifold (Millipore, Billerica, MA, USA).

[0073] Protein samples were loaded into a 96-well plate (AcroPrep Advance Filter Plates, 350 μL, Omega 30K MWCO). The proteins were reduced with 100 μL of 5 mM TCEP (37°C, 30 min), alkylated with 100 μL of 50 mM IAA (25°C, 30 min, in the dark), and digested with 6 mg of trypsin at 37°C for 18 hours. Subsequently, formic acid was added to a final concentration of 0.3% to lower the pH of the samples to 2-3 and stop the digestion. The peptide mixture was then desalted using a Sep Pak C-18 cartridge (Waters, USA), freeze-dried using a cold trap (CentriVap Cold Traps, LABCONCO), and stored at -80°C until use.

[0074] Prior to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis, dried peptide samples were dissolved in Buffer A (0.1% formic acid in HPLC water), and the total peptide concentration was measured using a UV / Vis spectrophotometer (NanoDrop One, Thermo Fisher Scientific). The wavelength was set to 280 nm, and the sample type option was set to "1 Abs = 1 mg / mL". Then, iRT standards provided by the iRT-Kit (Biognosys AG, Schlieren, Switzerland) were added to the samples at a volume of 1 / 10 to correct retention times according to vendor instructions (Reference: https: / / pubmed.ncbi.nlm.nih.gov / 22577012 / ). The total volume of each sample was 40 mg, dissolved in 40 mL. The injected 4 mL of sample was analyzed using a SCIEX TripleTOF 5600+ system mass spectrometer with the following LC-MS / MS setup.

[0075] For LC separation, a nanoLC 425 (Eksigent, Dublin, CA, USA) was used with an Eksigent microtrap C18 column (ChromXP C18CL, 5 μm, 120 Å) and an Eksigent column (C18-CL, 0.3 x 150 mm, particle size 3 μm, pore size 120 Å) as the analytical column, with the column temperature maintained at 40 °C. Samples were loaded into the trap column using buffer A at a flow rate of 10 μL / min. After 10 minutes, the peptide mixture was separated via a concentration gradient using buffer A and buffer B (0.1% formic acid in HPLC acetonitrile) at a flow rate of 5 μL / min for 57 minutes. Buffer B was changed to 3-25% for 38 minutes, 25-32% for 5 minutes, 32-80% for 2 minutes, maintained at 80% for 3 minutes, changed to 80-3% for 3 minutes, and maintained at 3% for 8 minutes. For individual samples, all mass spectrometry runs were operated in sequential window acquisition of all theoretical mass spectra (SWATH) modes using 100 variable windows in accordance with the SCIEX technical notes.

[0076] The SWATH parameters were set as follows: Lower m / z limit 400; m / z upper limit 1250; Window overlap (Da) 1.0; CES was set to 5 for the small window, 8 for the large window, and 10 for the largest window. MS2 spectra were collected in the 100–1500 m / z range for 2.5 ms in high sensitivity mode, and the total cycle time was 2.8 seconds. Other MS parameters were set as follows: Ion source gas 1 (GS1) 15 psi; Ion source gas 2 (GS2) 20 psi; Curtain gas (CUR) 30 psi; Temperature (TEM) 250 °C; Ion spray voltage floating (ISVF) 5500 V.

[0077] 3.2 Derivation of Biomarkers Associated with Lung Damage Caused by Humidifier Disinfectants and Humidifier Disinfectant Exposure

[0078] Using the quantitative information of the proteins collected in Example 3.1 above, biomarkers that appear to be associated with lung damage caused by humidifier disinfectants and exposure to humidifier disinfectants were derived.

[0079] Specifically, in addition, using quantitative information on a total of 280 proteins not associated with MARS-14 high abundant protein depletion that were detected in over 80% of plasma samples from 4 pediatric exposure groups (223 individuals) and a normal pediatric group (162 individuals), the importance of variables affecting the 5 groups via the XGBoost algorithm is 10 -45 The above 32 variables were selected. Secondly, training error was reduced by repeating 10-fold cross-validation 10,000 times using the 32 selected proteins. The parameters used were a maximum depth of 7 and a learning rate of 0.7. Using the Forward method of the XGBoost algorithm, 10 proteins with an absolute variable importance of 1 or greater were selected from the 32 proteins in descending order of coefficients. The selected proteins are shown in Table 3 below.

[0080] No Protein Protein full name 1 F13B Coagulation factor XIII B chain 2 APOF Apolipoprotein F 3 ITIH4 Inter-alpha-trypsin inhibitor heavy chain H4 4 FN1 Fibronectin 5 POTEF POTE ankyrin domain family member F 6 APOM Apolipoprotein M 7 APOL1 Apolipoprotein L1 8 SAA4 Serum amyloid A-4 protein 9 HPR Haptoglobin-related protein 10 APOB Apolipoprotein B-100

[0081] When comparing the quantitative levels of blood proteins with the humidifier disinfectant non-exposure control group, it was confirmed that F13B increased 1.5-fold, APOF decreased 3.8-fold, ITIH4 increased 1.3-fold, FN1 increased 1.7-fold, POTEF decreased 1.8-fold, APOM decreased 8.7-fold, APOL1 decreased 3.2-fold, SAA4 decreased 6.7-fold, HPR decreased 3.4-fold, and APOB decreased 5.5-fold. For all 10 proteins, it was confirmed that they were significant with a P-value of 0.05 or less in the T-test compared to the humidifier disinfectant non-exposure control group and the humidifier disinfectant exposure group.

[0083] Example 4. Model Construction Using Plasma Protein Data

[0084] A model was constructed to distinguish between the stages of lung damage caused by humidifier disinfectant exposure or exposure status using the quantitative information of proteins from the non-exposed control group and the exposed lung damage group (stages 1, 2, 3, and 4) collected in Example 3 above.

[0085] Specifically, using a determination tree structure, protein quantification information was treated as a variable, and an embedded method was applied in which variable selection is included within the model itself during the feature selection step.

[0086] A model was constructed using the XGBoost (eXtreme Gradient Boosting) technique, an evolved form of the Random Forest Algorithm. In the statistical model, the predictive scores for each group were analyzed for clinical association with clinical information such as the time of plasma collection, age, time of exposure to humidifier disinfectants, exposure substances and amounts, and family history to determine if there was a clinical association.

[0087] Using the quantitative information of 10 proteins extracted in Example 3.2 above, the pediatric samples of the non-exposed pediatric control group and the exposed lung injury pediatric group stages 1, 2, 3, and 4 were divided into three equal parts by group, and 2 / 3 of the information was used as a training set to construct the model, and the remaining 1 / 3 of the information was used as a test set to verify the completeness of the model to construct the model, and the model showing the best discriminative power was constructed using the XGBoost technique.

[0088] We reduced the training error by iterating 10,000 times using the Root Mean Square Deviation (RMSD), which the statistical model can distinguish. The parameters used were a maximum depth of 7 and a learning rate of 0.7.

[0089] Example 5. Model validation using plasma protein data

[0090] 5.1. Model Validation Using Plasma Protein Data in Children

[0091] Using a model composed of 10 protein panels listed in Table 3 (the model shown in Example 4), it was verified whether the non-exposure group and the exposed lung injury group (stages 1, 2, 3, and 4) of humidifier disinfectants could be distinguished.

[0092] Figure 1 is a graph showing a model that can distinguish between a normal group not exposed to humidifier disinfectants and a group with lung disease caused by humidifier disinfectants (stages 1, 2, 3, and 4) using an XGBoost analysis model containing 10 protein patterns in children.

[0093] As shown in Figure 1, a model consisting of 10 protein panels was found to be able to distinguish between the humidifier disinfectant non-exposure control group (mean predicted score 6.79) and four stepwise exposure groups (mean predicted scores 44.88, 47.71, 51.42, and 66.55) in children.

[0094] In addition, Figure 2 is a graph showing the results of verifying whether the normal group not exposed to humidifier disinfectants and the exposed group can be distinguished using the model shown in Example 4 in children.

[0095] As shown in Figure 2, when the specificity was fixed at 98.1%, the sensitivity was found to be 89.2% with a threshold value of 28.192 for the model's prediction score. The positive predictive value (accuracy in accurately predicting the exposed group as the exposed group) was 98.51%, and the negative predictive value (accuracy in accurately predicting the non-exposed group as the non-exposed group) was 86.89%. The Area under curve (AUC) of receiver operating characteristics (ROC), which indicates clinical utility, was found to be excellent at 0.9566.

[0096] The above results indicate that when using a model composed of 10 proteins to distinguish groups exposed to humidifier disinfectants, the group actually exposed to humidifier disinfectants can be distinguished from the group not exposed to humidifier disinfectants with 92.99% accuracy, regardless of the stage of lung disease. Therefore, this suggests that for pediatric applicants who actually used humidifier disinfectants but do not have direct evidence such as purchase receipts or related photos, a proteome test score of 28.192 or higher can be used as evidence of humidifier disinfectant use.

[0097] In addition, using the model shown in Example 4, we verified whether it is possible to distinguish between a normal group not exposed to humidifier disinfectants and a clinically almost certain Stage 1 lung disease caused by humidifier disinfectants in children, and the results are shown in Figure 3.

[0098] Figure 3 is a graph showing the results of verifying whether it is possible to distinguish between a normal group not exposed to humidifier disinfectants and a lung disease group with clinically almost certain stage 1 lung disease caused by humidifier disinfectants using the model shown in Example 4 in children.

[0099] As shown in Figure 3, when the threshold value of the model's prediction score was set to 25.551 while fixing the specificity at 95.7%, the model's sensitivity was found to be 97.6%. The positive predictive value was 90.90%, and the negative predictive value was 98.75%. The Area under curve (AUC) of the receiver operating characteristics (ROC) was found to be excellent at 0.996.

[0100] The above results indicate that the model composed of 10 proteins can distinguish the group with lung damage caused by humidifier disinfectants (Stage 1) from the group not exposed to humidifier disinfectants with an accuracy of 97.06%. Out of a total of 42 Stage 1 patients, 40 can be accurately classified as the Stage 1 group. Furthermore, the accuracy of the negative predictive value, which accurately predicts the non-exposed group as non-exposed, is 97.06%, confirming improved performance with a probability of approximately 3% where 4 out of a total of 162 non-exposed individuals may be misidentified as the exposed group.

[0102] 5.2. Model Validation Using Plasma Protein Data in Adults

[0103] Using a model composed of 10 protein panels listed in Table 3 (the model shown in Example 4), it was verified whether it is possible to distinguish between the non-exposure group and the exposure lung injury group (stages 1, 2, 3, and 4) of humidifier disinfectants in adults.

[0104] Figure 4 is a graph showing the results of verifying whether the normal group not exposed to humidifier disinfectants and the humidifier disinfectant lung disease group (stages 1, 2, 3, and 4) can be distinguished in adults using the model shown in Example 4.

[0105] As shown in Figure 4, the model shown in Example 4 was found to be able to distinguish between the non-exposure control group (average predicted score 14.41) and the four stepwise exposure groups (average predicted scores 44.64, 54.84, 50.33, and 61.88) in adults.

[0106] In addition, Figure 5 is a graph showing the results of verifying whether the normal group not exposed to humidifier disinfectants and the exposed group can be distinguished in adults using the model shown in Example 4.

[0107] As shown in Figure 5, when the specificity was fixed at 98%, the sensitivity was found to be 85.1% with a threshold value of 36.977 for the model's prediction score. The positive predictive value (accuracy in accurately predicting the exposed group as the exposed group) was 97.56%, and the negative predictive value (accuracy in accurately predicting the non-exposed group as the non-exposed group) was 87.5%. The Area under curve (AUC) of receiver operating characteristics (ROC), which indicates clinical utility, was found to be excellent at 0.9736.

[0108] The above results indicate that when using the model shown in Example 4 to distinguish groups exposed to humidifier disinfectants, the adult group actually exposed to humidifier disinfectants can be distinguished from the group not exposed to humidifier disinfectants with an accuracy of 91.75%, regardless of the stage of lung disease. Therefore, this suggests that for applicants who actually used humidifier disinfectants but do not have direct evidence such as purchase receipts or related photos, a proteome test score of 39.977 or higher can be used as evidence of humidifier disinfectant use.

[0109] In addition, using the model shown in Example 4, we verified whether it is possible to distinguish between a normal group not exposed to humidifier disinfectants and a patient group with clinically almost certain stage 1 lung disease caused by humidifier disinfectants in adults, and the results are shown in Figure 6.

[0110] Figure 6 is a graph showing the results of verifying whether it is possible to distinguish between a normal group not exposed to humidifier disinfectants and a clinically almost certain stage 1 lung disease caused by humidifier disinfectants in adults using the model shown in Example 4.

[0111] As shown in Figure 6, when the threshold value of the model's prediction score was set to 36.299 while fixing the specificity at 98%, the model's sensitivity was found to be 100%. The positive predictive value was 93.33%, and the negative predictive value was 100%. The Area under curve (AUC) of the receiver operating characteristics (ROC) was found to be excellent at 0.9986.

[0112] The above results indicate that the model composed of 10 proteins can distinguish the group with lung damage caused by humidifier disinfectants (Stage 1) from the group not exposed to humidifier disinfectants with an accuracy of 98.44%. It can accurately classify 13 out of a total of 14 Stage 1 patients into the Stage 1 group. Furthermore, the negative predictive value, which accurately predicts the non-exposed group as non-exposed, was 100%, confirming that the model demonstrates the ability to identify all 50 non-exposed individuals as non-exposed.

Claims

Claim 1 A diagnostic composition capable of predicting lung damage caused by humidifier disinfectants or predicting exposure to humidifier disinfectant components, comprising a preparation capable of measuring the expression level of one or more proteins or genes selected from the group consisting of F13B, ITIH4, POTEF, APOM, APOL1, and HPR. Claim 2 The composition of claim 1, wherein the diagnostic composition further comprises a preparation capable of measuring the expression level of one or more proteins or genes selected from the group consisting of APOF, FN1, SAA4, and APOB, and is capable of predicting lung damage caused by a humidifier disinfectant or predicting exposure to a humidifier disinfectant component. Claim 3 Claim 1, wherein the diagnostic composition is capable of predicting lung damage caused by humidifier disinfectants in children or adults and predicting whether there has been exposure to humidifier disinfectant components. Claim 4 A diagnostic composition according to claim 1, comprising one or more selected from the group consisting of antibodies that specifically recognize the protein, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers. Claim 5 The formulation capable of measuring the gene expression level according to claim 1 is a diagnostic composition comprising one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to said gene. Claim 6 A kit for predicting lung damage caused by a humidifier disinfectant or predicting exposure to a humidifier disinfectant component, comprising any one of the compositions of claims 1 to 5. Claim 7 A method for providing information for predicting lung damage caused by a humidifier disinfectant or predicting exposure to a humidifier disinfectant component, comprising: a step of measuring the expression level of one or more proteins or genes selected from a group consisting of F13B, ITIH4, POTEF, APOM, APOL1, and HPR in a biological sample isolated from a target individual; a step of generating a score by inputting a specific weight into an algorithm based on the expression level of said protein or gene; and a step of providing information analyzed as indicating a high probability of lung damage caused by a humidifier disinfectant or exposure to a humidifier disinfectant component when the generated score is higher than that of a control group not exposed to the humidifier disinfectant. Claim 8 A method for providing information for predicting lung damage caused by a humidifier disinfectant or predicting exposure to a humidifier disinfectant component, wherein the step of measuring the expression level of a protein or gene in the separated biological sample comprises measuring the expression level of one or more proteins or genes selected from the group consisting of APOF, FN1, SAA4, and APOB. Claim 9 A method according to claim 7, wherein the biological sample is selected from the group consisting of blood, serum and plasma.

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