Hemoglobin-related gene for diagnosing immune status and use thereof

Abstract

The present invention relates to a composition for diagnosing immunodeficiency or a method for providing information for diagnosis thereof. By using the composition and the method according to one aspect, it is possible to simply and effectively diagnose immune-deficient conditions such as immunosuppression or immune paralysis.

Description

Hemoglobin-related genes and their uses for diagnosing immune status

[0001] The present invention relates to a genetic biomarker for diagnosing immune status.

[0002] Sepsis is a condition in which bacteria or other pathogens invade the bloodstream, causing serious systemic reactions such as toxicity or acute inflammation. When viruses or bacteria first infect the bloodstream due to an infectious disease or trauma, these bacteria multiply within the bloodstream, triggering a systemic inflammatory response including high fever, increased white blood cells, and low blood pressure. In healthy individuals with strong immune systems, white blood cells can fight off these invading bacteria. However, sepsis is more likely to develop in those with weakened immune systems due to the elderly, immunosuppressants such as those receiving chemotherapy, liver disease, alcoholism, or malnutrition, as well as in newborns.

[0003] Sepsis can have a good prognosis if treated promptly with large amounts of fluids and antibiotics in the early stages. However, there is currently no simple and easy way to assess the immune status of sepsis patients worldwide. Assessing the immune status of a sepsis patient's immune cells requires a complex process that takes at least six hours, and while this can be done in the laboratory, it is extremely difficult to implement in a hospital setting.

[0004] Meanwhile, the inventors of the present invention discovered that transcriptomics can be used to determine the status of immune cells in sepsis patients, including immunohyperactivity / immunosuppression. Specifically, by assessing the expression levels of genes associated with hemoglobin, the inventors discovered that immunodeficiency, such as immunosuppression or immunoparalysis, associated with diseases like sepsis or cancer, can be diagnosed with remarkable ease, leading to the completion of the present invention.

[0005] One aspect is to provide a biomarker composition for diagnosing immunodeficiency, which comprises a hemoglobin-related gene in an immune cell.

[0006] Another aspect is to provide a composition for diagnosing immunodeficiency comprising a preparation capable of measuring the level of expression or activity of a hemoglobin-related gene in an immune cell.

[0007] Another aspect is to provide a kit for diagnosing immunodeficiency comprising the diagnostic composition.

[0008] Another aspect provides a method for providing information for diagnosing an immunodeficiency, comprising the steps of: measuring the expression or activity level of a hemoglobin-related gene in an immune cell in a sample isolated from an individual; and comparing the expression or activity level with the expression or activity level of a normal control.

[0009] One aspect provides a biomarker composition for diagnosing immunodeficiency, which includes a hemoglobin-related gene in an immune cell.

[0010] Another aspect provides a composition for diagnosing immunodeficiency comprising a formulation capable of measuring the level of expression or activity of a hemoglobin-related gene in immune cells.

[0011] In one specific example, the hemoglobin-related genes are Hpgds (hematopoietic prostaglandin D synthase), Gzmm (Granzyme M), Mylk2 (myosin light chain kinase 2), Car1 (carbonic anhydrase 1), Cd52, Akr7a3 (Aldo-keto reductase family 7 member A3), Dnajb13 (DnaJ heat shock protein family (Hsp40) member B13), Alox15 (arachidonate 15-lipoxygenase), Hbe1 (hemoglobin subunit epsilon 1), Gsta4 (glutathione S-transferase alpha 4), Epb42 (erythrocyte membrane protein band 4.2), Ahsp (alpha hemoglobin stabilizing protein), Rhd (Rh blood group D antigen), Hbb (hemoglobin subunit beta), Hba3 (hemoglobin alpha, adult chain 3), Alas2 (5'-aminolevulinate synthase 2), Hbb-b1 (hemoglobin, beta adult major chain), Hbq1b (hemoglobin subunit theta 1B), Hbz (hemoglobin subunit zeta), Hba2 (hemoglobin alpha, adult chain 2), Hba1 (hemoglobin alpha, adult chain 1), GATA-1 (Gata binding protein 1), KLF4 (Kruppel-like factor 4), KLF1 (Kruppel-like factor 1), and KLF2 (Kruppel-like factor 2).

[0012] The term “immunodeficiency” in this specification refers to a general term for diseases caused by dysfunction of immune system components, and means a state in which the ability of the immune system to fight diseases such as infectious diseases and cancer is reduced or completely absent.

[0013] In one specific example, the immunodeficiency may be immunosuppression or immune paralysis.

[0014] In one specific example, the immunodeficiency may be accompanied by one or more of cancer, sepsis, and severe trauma.

[0015] The term “diagnosis” as used herein means confirming the presence or characteristics of a pathological condition, and may refer to determining the susceptibility of an object to a specific disease or condition, determining whether an object has a specific disease or condition, determining the prognosis of an object with a specific disease or condition, or monitoring the same. “Prognosis” means determining whether a subject will have treatment success, survival, recurrence, metastasis, drug responsiveness, resistance, etc. after treatment. In other words, it means the expectation of a medical outcome (e.g., long-term survival possibility, disease-free survival rate, etc.), and includes a positive prognosis (positive prognosis) or a negative prognosis (negative prognosis), and the negative prognosis includes the progression or mortality of the disease, such as recurrence, metastasis, drug resistance, etc., and the positive prognosis includes the improvement or stabilization of the disease, such as the remission of the disease, or tumor regression, etc. For the purposes of the present invention, diagnosis within the present specification may mean diagnosis of immunodeficiency.

[0016] 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.

[0017] The present invention can provide a composition comprising the above-mentioned biomarker that changes according to the onset of a disease and / or an agent capable of measuring the degree of expression or activity of the above-mentioned biomarker. That is, the expression or activity of a hemoglobin-related gene in the present specification can mean the mRNA expression or activity level of the hemoglobin-related gene or the expression or activity level of the hemoglobin protein, and measuring the expression or activity level of the hemoglobin-related gene can mean measuring the degree of expression or activity of the above-mentioned biomarker that changes according to the onset of a disease. For example, it can mean measuring the degree of expression or activity of a marker protein with an agent capable of measuring the level of the protein, and it can mean measuring the degree of expression or activity of a marker gene with an agent capable of measuring the level of the gene.

[0018] In one specific example, the agent for measuring the level of the gene may be an agent for measuring the level of mRNA of the gene, and 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.

[0019] The measurement thereof may be performed by a method selected from the group consisting of, but not limited to, polymerase chain reaction (PCR), real-time polymerase chain reaction (qPCR), reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, RNase and S1 nuclease protection assay, in situ hybridization, nucleic acid microarray, northern blotting, or DNA chip.

[0020] 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.

[0021] 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.

[0022] The above primers or probes can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of a natural nucleotide, and modifications between nucleotides, such as modification with uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

[0023] Suitable conditions for hybridizing probes with cDNA molecules can be determined through a series of optimization procedures. These procedures are implemented in a series of steps by those skilled in the art to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing times, buffer components, and their pH and ionic strength depend on various factors such as the length and GC content of the probe and the target nucleotide sequence. Detailed hybridization conditions can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and M. L. M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc., NY (1999). For example, among the above stringency conditions, high stringency conditions mean hybridization at 65°C in 0.5 M NaHPO4, 7% SDS (sodium dodecylsulfate), 1 mM EDTA, and washing at 68°C in 0.1 x SSC (standard saline citrate) / 0.1% SDS. Alternatively, high stringency conditions mean washing at 48°C in 6 x SSC / 0.05% sodium pyrophosphate. Low stringency conditions mean washing at 42°C in 0.2 x SSC / 0.1% SDS, for example.

[0024] The above PNA stands for peptide nucleic acid (PNA), and refers to an artificially synthesized polymer similar to DNA or RNA. It is a DNA-like structure in which the nucleic acid bases are linked by peptide bonds rather than phosphate bonds. While DNA has a phosphate-ribose sugar backbone, PNA has a repeating N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which can significantly increase binding affinity and stability to the DNA or RNA of the target sequence.

[0025] 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.

[0026] In one embodiment, the agent for measuring the level of the protein may comprise one or more selected from the group consisting of antibodies, antibody fragments, interacting proteins, oligopeptides, ligands, nanoparticles, aptamers, avidity multimers, and peptidomimetics that specifically bind to the protein or a peptide fragment thereof.

[0027] The measurement thereof can be performed by a method 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), but is not limited thereto.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Since information on the marker protein according to the present invention or the gene encoding it is known in the art, a person skilled in the art can design a preparation for measuring the level of the protein or the gene encoding it based on this.

[0034]

[0035] Another aspect provides a kit for diagnosing immunodeficiency comprising the composition.

[0036] 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.

[0037] The kit may further comprise one or more other component compositions, solutions or devices suitable for the analytical method.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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)).

[0045] In one specific example, the kit may include a preparation, a device, and a computer having an algorithm built in for measuring the level of mRNA of the protein or a gene encoding the protein, and may relate to a kit that associates the result of measuring the level of the marker with the prediction of immunodeficiency through the algorithm.

[0046]

[0047] Another aspect provides a method for providing information for diagnosing immunodeficiency, comprising the steps of measuring the expression or activity level of a hemoglobin-related gene in immune cells in a sample isolated from an individual; and comparing the expression or activity level with the expression or activity level of a normal control.

[0048] The term “subject” or “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.

[0049] The term “normal individual” or “normal control group” in this specification may refer to a general individual who has not developed immunodeficiency, a non-immunodeficiency patient group, a non-patient group, etc.

[0050] The term “sample” as used herein means any material, biological fluid, tissue or cell obtained from or derived from a subject who is at risk for, has developed or is suspected of developing an immunodeficiency.

[0051] In one specific example, the sample may be at least one selected from the group consisting of whole blood, white blood cells, peripheral blood mononuclear cells, white blood cell buffy coat, blood, including plasma and serum, sputum, tears, mucus, nasal fluid, nasal aspirate, breath, urine, semen, saliva, peritoneal lavage, pelvic fluid, cystic 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.

[0052] In one specific example, the sample may be blood, plasma, blood cells, serum, peripheral blood mononuclear cells, spleen cells, or a combination thereof.

[0053] In one specific example, the sample may be an immune cell, and specifically, an immune cell of tissue, spleen or blood.

[0054] According to one specific example, the method of the present invention can perform appropriate processing (e.g., removal of abundant protein, peptidization, etc.) on a sample 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 in a sample separated from a subject.

[0055] In one specific example, the method may further include a step of determining that the subject has an immunodeficiency if the expression or activity of the hemoglobin-related gene is reduced compared to a normal control group.

[0056] In one embodiment, the step of measuring the expression or activity level may include the step of measuring the mRNA level of the hemoglobin-associated gene.

[0057] In one specific example, the measurement of the mRNA level can be performed by a method selected from the group consisting of polymerase chain reaction (PCR), real-time polymerase chain reaction (qPCR), reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, RNase and S1 nuclease protection assay, in situ hybridization, nucleic acid microarray, northern blotting, or DNA chip, but is not limited thereto.

[0058] In one embodiment, the step of measuring the expression or activity level may include the step of measuring the protein level of the hemoglobin-associated gene.

[0059] In one specific example, the measurement of the protein level can be performed by a method 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), but is not limited thereto.

[0060]

[0061] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0062] A composition for diagnosing immunodeficiency according to an aspect or a method for providing information for diagnosing the same relates to a hemoglobin-related gene, which is a disease-specific biomarker that changes in an immunodeficiency patient, and by measuring and comparing the level of the gene, an immunodeficiency state such as immunosuppression or immunoparalysis can be simply and effectively diagnosed.

[0063] Figure 1 is a schematic diagram of an experiment to select biomarkers for diagnosing immune levels.

[0064] Figure 2 is a graph showing the PCA analysis results for each group (Sham, Sepsis_6h, Sepsis_24h, Sepsis_5d).

[0065] Figure 3 is a graph showing the results of DEG analysis between each group (Sham, Sepsis_6h, Sepsis_24h, Sepsis_5d): FDR < 0.05, FC > 2, x & y axis: Log2(CPM + 1)

[0066] Figure 4 is a diagram analyzing genes with high or low expression patterns in the Sepsis_24h group compared to other groups (Sham, Sepsis_6h, Sepsis_5d) to select biomarkers.

[0067] Figure 5 is a diagram of the Hbe1 gene network: 24h low peak genes= Mylk2, Cd52, Car1, Hpgds, Hbe1, Alox15, Gsta4, Dnajb13, Akr7a3, Gzmm; Genes, more founded= Cacng1, Klhl41, Myh3, Hba-a3, Hbb, Cdc25b, Alas2, Ahsp, Epb42, Kel, Hemgn, Slc4a1, Rhd; Mild 24h low peak genes= Hba-a3, Hbb, Alas2, Cdc25b, Kel, Rhd.

[0068] The following examples are provided for more detailed description. However, these examples are provided solely to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0069]

[0070] Example 1. Preparation of experimental animals in which an immunosuppressive response is induced.

[0071] First, splenocyte samples, one of the immune cell populations, were obtained from mouse models induced with immunodeficiency and control mice.

[0072] Specifically, a cecal slurry model, a globally recognized intraperitoneal sepsis model, was created using male Sprague-Dawley rats weighing 270-300 g. Sepsis was induced by collecting feces from donor rats, dividing the collected feces into a predetermined amount, and injecting the collected feces into the abdominal cavity of the rats to be induced. Specifically, the donor rats were anesthetized with an intramuscular injection of Zoletil (50 mg / kg) and xylazine (10 mg / kg). A midline laparotomy was performed, and the cecum was protruded. A 0.5 cm incision was made on the anti-serosal surface of the cecum, and the cecum was compressed to drain the feces. The feces were collected, weighed, and diluted 1:3 with 5% glucose saline. The sepsis-induced rats were anesthetized in the same manner, and a 10.5 cm midline laparotomy was performed, and the fecal slurry was administered intraperitoneally. The fecal slurry was vortexed to obtain a homogeneous suspension before intraperitoneal administration. The volume of fecal slurry administered to each animal was adjusted based on the body weight of the rats induced with sepsis. After sepsis induction, treatment for sepsis included subcutaneous fluid resuscitation (30 mL / kg 5% glucose saline) and antibiotics, including imipenem administered subcutaneously at a dose of 25 mg / kg twice daily for 2 days.

[0073] Blood and spleens were collected from mice for metabolomic analysis at 6, 24, and 5 days after sepsis induction. Peripheral blood mononuclear cells (PBMCs) were obtained from the blood, and splenocytes were obtained from the spleen. In the control group, the same samples were collected from non-sepsis-induced mice (Sham) at 6, 24, and 5 days for metabolomic analysis. A brief schematic of the experiment is shown in Figure 1.

[0074]

[0075] Example 2. RNA extraction and sequencing

[0076] The spleen cells collected in Example 1 were isolated, and total RNA was extracted from each sample by treating it with Trizol reagent (Invitrogen). Quality control and quantification were performed using a Bioanalyzer 2100 system (Agilent Technologies, USA) and a Nanodrop ND-2000 Spectrophotometer (Thermo Scientific, USA). For RNA sequencing (RNA-seq), the NEBNext Ultra II Directional RNA-Seq Kit (NEW ENGLAND BioLabs, Inc., UK) was used according to the manufacturer's instructions to construct a library from total RNA. Briefly, the isolated mRNA was used to ligate adapters, and then cDNA was synthesized using adapter-specific primers and reverse transcriptase. After performing PCR for library amplification, high-throughput sequencing was performed using TapeStation HS D1000 Screen Tape (Agilent Technologies, USA), StepOne Real-Time PCR System (Life Technologies, Inc., USA), and NovaSeq 6000 (Illumina, Inc., USA) with paired-end 101 base pair reads.

[0077]

[0078] Example 3. Genome-wide transcriptome analysis

[0079] 3.1 Transcriptome analysis method

[0080] The content of the transcript obtained in Example 2 was analyzed, and then the analyzed transcript was used as raw data to derive a group of biomarker candidates related to immune suppression through a public database.

[0081] All raw sequence reads were preprocessed using Trimmomatic (version 0.39) to remove low-quality bases and adapter sequences. The mouse reference genome (Rnor_6.0), gene model annotation files, and genome indexes constructed using hisat2-build were obtained from the Ensembl database (http: / www.ensembl.org). The trimmed paired-end reads were aligned to the reference genome using HISAT2 (v2.2.1) with default parameters. The binary alignment map (BAM) files generated by the HISAT2 aligner were further processed with StringTie (v2.2.1) and prepDE, and transcript abundance was quantified using transcripts per kilobase million (TPM) and counts per million mapped reads (CPM). CPM quantification and differential expression analysis were performed using the edgeR package (v3.38.4), and differentially expressed genes (DEGs) were analyzed if they had read count > 10, false discovery rate q-value (FDR) < 0.05, and log 2-fold change > 1 in at least one sample.

[0082]

[0083] 3.2 PCA (principal component analysis) analysis results

[0084] The results of PCA analysis to check the similarity after calculating the expression level by analyzing the acquired transcripts are shown in Figure 2.

[0085] As shown in Fig. 2, the transcript expression patterns were similar between the same groups, the transcript expression patterns between the Sham and Sepsis_5d groups were similar, and the transcript expression patterns between the Sepsis_6h group and the Sepsis_24h group were similar.

[0086]

[0087] 3.3 DEG (differentially expressed genes) analysis results

[0088] The results of analyzing genes (DEGs) that showed different expression patterns between each group are shown in Figures 3 to 5 and Table 1.

[0089] Figure 3 is a graph showing the results of DEG analysis between each group (Sham, Sepsis_6h, Sepsis_24h, Sepsis_5d): FDR < 0.05, FC > 2, x & y axis: Log2(CPM + 1)

[0090] As shown in Figure 3, when examining the data that deviate from the line, the transcriptome expression patterns between the Sham and Sepsis_5d groups were similar, and the transcriptome expression patterns between the Sepsis_6h and Sepsis_24h groups were similar. The more data that deviate from the line, the more genes that show differences between the two groups.

[0091] As shown in Fig. 4, when analyzing genes with high or low expression patterns in the Sepsis_24h group compared to other groups (Sham, Sepsis_6h, Sepsis_5d), 64 24h high peak genes and 55 24h low peak genes were analyzed.

[0092] Based on the above statistical analysis results, hemoglobin-related genes were analyzed, and Table 1 shows the DEG analysis results compared to the Sham group, and Table 2 shows the average expression levels of genes.

[0093]

[0094]

[0095]

[0096] Gene nameEnsembl IDAverage expression level (CPM)ShamSepsis_6hSepsis_24hSepsis_5dHpgdsENSRNOG00000006583216.2764.7218.90101.15GzmmENSRNOG00000030530113.9338.9418.0847.81Mylk2ENSRNOG0000000823541.4810.885.8336.60Car1ENSRNOG00000010698165.1949.0018.62295.90Cd52ENSRNOG0000001540343.7316.098.4936.30Akr7a3ENSRNOG000000178999.234.012.228.80Dnajb13ENSRNOG0000001797511.245.221.718.19Alox15ENSRNOG00000019183256.4478.9039.19313.08Hbe1ENSRNOG000000292862.020.950.341.95Gsta4ENSRNOG0000003044931.4611.875.4432.75Epb42ENSRNOG0000001164999.7642.6344.7589.83KelENSRNOG0000001568248.4337.5425.0154.02HemgnENSRNOG00000009436126.95150.6786.11145.92AhspENSRNOG00000020165814.01189.01194.82773.44Slc4a1ENSRNOG00000020951882.62910.85591.24849.55Myh3ENSRNOG000000462760.430.360.220.17Cdc25bENSRNOG00000021248327.58181.74112.00207.71RhdENSRNOG00000017130168.7080.7567.37175.65HbbENSRNOG0000005810543758.2013645.7310670.8723355.12Hba-a3ENSRNOG00000045989799.45280.31170.82262.99Alas2ENSRNOG00000000167885.47390.06244.88579.97Cacng1ENSRNOG00000003245Klhl41ENSRNOG00000007461Hbb-b1ENSRNOG0000004709813,230.024,680.703,345.228,418.82Hbq1bENSRNOG 0000002811420.5210.026.8720.45HbzENSRNOG0000002053625.140.650.4 017.80Hbg1ENSRNOG000000308790.580.070.160.29Hba2ENSRNOG00000047 32131,496.3210,542.196,192.6016,300.07Hba1ENSRNOG0000002988654, 774.7719,103.5712,019.6430,324.82GATA-1ENSRNOG0000004766321.310 .37.022.8KLF4ENSRNOG0000001629944.795.845.122.5KLF1ENSRNOG0000000344391.225.924.6112.1KLF2ENSRNOG00000014205132.055.464.161.7.

[0097]

[0098] As shown in Tables 1 and 2, the expression levels of hemoglobin-related genes decreased for up to 24 hours and then recovered.

[0099] Previous studies have shown that sepsis-induced mice show a decrease in the immune status of immune cells starting from 6 hours, show the lowest immunosuppression at 24 hours, and enter the recovery phase from 5 days. (Int J Mol Sci. 2022 Jun 13;23(12):6581. doi: 10.3390 / ijms23126581.Serial Change of Endotoxin Tolerance in a Polymicrobial Sepsis Model) That is, if we summarize the above results, hemoglobin-related genes can play a very important role in the survival of immune cells and can be useful as biomarkers for diagnosing immunodeficiency.

[0100]

[0101] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A biomarker composition for diagnosing immunodeficiency comprising a hemoglobin-related gene in an immune cell.

2. A composition for diagnosing immunodeficiency, comprising a preparation capable of measuring the expression or activity level of a hemoglobin-related gene in immune cells.

3. A composition according to claim 2, wherein the preparation comprises at least one selected from the group consisting of a primer, a probe, a peptide nucleic acid (PNA), and an antisense nucleotide that specifically binds to the hemoglobin-related gene.

4. A composition according to claim 2, wherein the preparation comprises at least one selected from the group consisting of antibodies, antibody fragments, interacting proteins, oligopeptides, ligands, nanoparticles, aptamers, avidity multimers, and peptidomimetics that specifically bind to the protein of the hemoglobin-related gene or a peptide fragment thereof.

5. A composition according to any one of claims 1 to 4, wherein the hemoglobin-related gene comprises at least one selected from the group consisting of Hpgds, Gzmm, Mylk2, Car1, Cd52, Akr7a3, Dnajb13, Alox15, Hbe1, Gsta4, Epb42, Ahsp, Rhd, Hbb, Hba3, Alas2, Hbb-b1, Hbq1, Hbz, Hba2, Hba1, GATA-1, KLF4, KLF1, and KLF2.

6. A composition according to any one of claims 1 to 4, wherein the immunodeficiency is immunosuppression or immune paralysis.

7. A composition according to any one of claims 1 to 4, wherein the immunodeficiency is accompanied by one or more diseases selected from the group consisting of cancer, sepsis, and severe trauma.

8. A kit for diagnosing immunodeficiency comprising the composition of any one of claims 2 to 4.

9. A step of measuring the expression or activity level of hemoglobin-related genes in immune cells in a sample isolated from an individual; and A method for providing information for diagnosing immunodeficiency, comprising a step of comparing the expression or activity level with the expression or activity level of a normal control.

10. A method according to claim 9, further comprising a step of determining that the subject has immunodeficiency when the expression of the hemoglobin-related gene is reduced compared to a normal control group.

11. In claim 9, the step of measuring the expression or activity level includes the step of measuring the mRNA level of the hemoglobin-related gene, A method wherein the measurement of mRNA levels is performed by at least one method selected from the group consisting of polymerase chain reaction (PCR), real-time polymerase chain reaction (qPCR), reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, RNase and S1 nuclease protection assay, in situ hybridization, nucleic acid microarray, northern blotting, or DNA chip.

12. In claim 9, the step of measuring the expression or activity level includes the step of measuring the protein level of the hemoglobin-related gene, A method wherein the measurement of protein levels is performed by at least one selected from the group consisting of protein mass spectrometry, protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, aukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry), Western blot, and ELISA (Enzyme Linked Immunosorbent Assay).

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