Methods for detecting diabetic neuropathy

Abstract

To provide a method for detecting diabetic neuropathy using protein in a biological sample.SOLUTION: A detection method for diabetic neuropathy in a subject includes a step of measuring at least one expression level of protein selected from LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, CCN5, FMNL1, SCGN, CXCL14, BACH1, LY6D, CCL24, SPINT2, CDH15, CTSC, CCL13, NOS1, SCARA5, CCL11, ACVRL1, CD276, SUSD2, TMSB10, NT5C3A, TNFRSF6B and SPINK1 regarding a biological sample obtained from the subject.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for detecting diabetic neuropathy using a biomarker. Background Art

[0002] Currently, there are approximately 400 million people with diabetes worldwide, and it is said that about 12% of global health expenditure is spent on diabetes or its complications. It is widely recognized that the danger of diabetes lies in its complications, and diabetic neuropathy is one of the major complications. Diabetic neuropathy is a nerve length-dependent neuropathy that progresses symmetrically from the distal extremities. In the early stage, it presents pain and numbness, but gradually leads to decreased sensation and impaired blood flow. In the worst case, gangrene results in amputation of the extremities, which significantly reduces patients' quality of life (QOL).

[0003] It is known that as diabetic neuropathy progresses, the incidence of foot lesions, ischemic cerebrovascular disease, and ischemic heart disease increases. Furthermore, it is known that while advanced cases are difficult to cure, early-stage cases can be improved. Based on the above, early detection and intervention are critical for diabetic neuropathy.

[0004] On the other hand, there are challenges in the diagnosis of diabetic neuropathy. That is, nerve conduction studies are required for definitive diagnosis of diabetic neuropathy, but this method has several problems: it requires dedicated equipment, skillful operation, takes time depending on the number of target nerves to be tested, and involves electrical stimulation that causes pain or discomfort (Non-Patent Documents 1 and 2). In addition, half of patients with diabetic neuropathy do not have subjective symptoms, so by the time the condition is noticed, gangrene has often progressed and amputation of the leg is unavoidable in many cases.

[0005] Based on the above, establishment and widespread use of simple and reliable nerve monitoring technology are required, but no biomarker capable of detecting diabetic neuropathy has been found to date. Prior Art Documents Non-Patent Documents

[0006] [Non-Patent Document 1] Clinical Neurophysiology. 2018, 46: 71-77 [Non-Patent Document 2] Various scores for diabetic neuropathy. 2013, Diabetic Neuropathy (Nakayama Shoten); 37-48 [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention relates to providing a method for detecting diabetic neuropathy using biomarkers. [Means for solving the problem]

[0008] The inventors of this invention have discovered that among diabetic patients, there are proteins whose expression levels in the blood of patients who have developed neuropathy and those who have not differed significantly between the two groups, and that this can be used as an indicator to detect diabetic neuropathy.

[0009] In other words, the present invention relates to the following 1) to 3). 1) A method for detecting diabetic neuropathy in a subject, comprising the step of measuring the expression level of at least one protein selected from LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, CCN5, FMNL1, SCGN, CXCL14, BACH1, LY6D, CCL24, SPINT2, CDH15, CTSC, CCL13, NOS1, SCARA5, CCL11, ACVRL1, CD276, SUSD2, TMSB10, NT5C3A, TNFRSF6B, and SPINK1 in a biological sample taken from the subject. 2) A test kit for detecting diabetic neuropathy used in the method of 1) above, comprising a molecule that binds to the protein or an oligonucleotide that specifically hybridizes with the gene encoding the protein. 3) A marker for detecting diabetic neuropathy, comprising at least one protein selected from LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, CCN5, FMNL1, SCGN, CXCL14, BACH1, LY6D, CCL24, SPINT2, CDH15, CTSC, CCL13, NOS1, SCARA5, CCL11, ACVRL1, CD276, SUSD2, TMSB10, NT5C3A, TNFRSF6B, and SPINK1. [Effects of the Invention]

[0010] According to the present invention, diabetic neuropathy can be detected simply and non-invasively with high accuracy, sensitivity, and specificity. [Brief explanation of the drawing]

[0011] [Figure 1] Comparison of plasma proteomes between diabetic patients with neuropathy (DPN) and diabetic patients without neuropathy (DM). [Figure 2] Passive operating characteristic curves (ROCs) for diabetic neuropathy of 29 blood proteins. [Modes for carrying out the invention]

[0012] All patent, non-patent, and other publications cited herein are incorporated herein by reference in their entirety.

[0013] In this invention, the terms "nucleic acid" or "polynucleotide" mean DNA or RNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and RNA includes total RNA, mRNA, rRNA, tRNA, non-coding RNA, and synthetic RNA.

[0014] In this invention, "gene" means a double-stranded DNA including human genomic DNA, as well as single-stranded DNA (positive strand) including cDNA, single-stranded DNA (complementary strand) having a sequence complementary to the positive strand, and fragments thereof, in which the sequence information of the bases constituting the DNA contains some kind of biological information. Furthermore, the term "gene" in question encompasses not only genes represented by a specific base sequence, but also nucleic acids encoding their homologs (i.e., orthologs), variants such as genetic polymorphisms, and derivatives.

[0015] In this invention, "diabetic neuropathy" refers to nerve damage that appears after the onset of diabetes. Diabetic neuropathy is divided into distal symmetrical polyneuropathy and focal mononeuropathy, but the diabetic neuropathy of this invention includes both. Distal symmetrical polyneuropathy is divided into sensory-motor neuropathy and autonomic neuropathy. In sensory-motor neuropathy, a decrease in nerve conduction velocity is observed from the early stages of onset by nerve conduction studies. Sensory abnormalities such as spontaneous pain, numbness, paresthesia, and hypoesthesia appear in the extremities of the lower limbs, as well as motor abnormalities such as muscle weakness, muscle atrophy, and impaired balance. As the symptoms progress upward, symptoms also appear in the extremities of the upper limbs. Furthermore, eye movements and facial movements are also impaired. Autonomic neuropathy presents with a variety of pathological conditions, including pupillary dysfunction, orthostatic hypotension, cardiac nerve disorders (sudden death, painless myocardial infarction), abnormal sweating, gastrointestinal motility disorders (constipation, diarrhea), bladder dysfunction, and erectile dysfunction. Focal mononeuropathy includes cranial nerve disorders (especially extraocular muscle palsy), nerve disorders of the trunk and limbs, and diabetic muscle atrophy (lumbosacral root plexus neuropathy).

[0016] Diabetic neuropathy is diagnosed when two or more of the following criteria are met: 1) lower extremity sensory abnormalities (pain, numbness, hypoesthesia), 2) decreased or absent bilateral Achilles tendon reflexes, and 3) decreased vibration sensation in both medial malleoli (loss of vibration sensation of C128 tuning fork within 10 seconds). Alternatively, even if these criteria are not met, if nerve conduction studies show abnormal values ​​in both feet, neuropathy is considered present (Diabetic Neuropathy Study Group, Simplified Diagnostic Criteria for Diabetic Polyneuropathy (Minor Revision), Peripheral Nerves 23:109-111, 2012).

[0017] In this invention, "detection of diabetic neuropathy" means clarifying the presence or absence of neuropathy in diabetic patients. In this invention, the term "detection" may also be replaced with terms such as examination, measurement, determination, evaluation, or evaluation support. In this specification, the terms "determination" or "evaluation" do not include determination or evaluation by a physician.

[0018] As shown in the examples described later, 43 men and women with diabetes mellitus were diagnosed for the presence or absence of diabetic neuropathy using the above diagnostic criteria and classified into diabetic patients with neuropathy (DPN) and diabetic patients without neuropathy (DM). Plasma proteome analysis was performed on each group, and a total of 732 proteins were quantified, consisting of an inflammatory panel (368 types) and a neurology panel (367 types) (due to 3 molecule overlaps between the two panels). As a result, the following 29 proteins were identified that showed significant differences in blood concentration between the DPN and DM groups (Figure 1). Of these, only LILRA2 was a protein whose expression decreased in DPN, while the others were proteins whose expression increased in DPN. LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, CCN5, FMNL1, SCGN, CXCL14, BACH1, LY6D, CCL24, SPI NT2, CDH15, CTSC, CCL13, NOS1, SCARA5, CCL11, ACVRL1, CD276, SUSD2, TMSB10, NT5C3A, TNFRSF6B, SPINK1. Therefore, these 29 types of proteins (hereinafter also referred to as "target proteins") are useful as diabetic neuropathy markers for detecting diabetic neuropathy. Among these, from the viewpoint of detection accuracy, it is preferable to use one or more selected from LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, SPINK1 and CCN5, and it is more preferable to use one or more selected from LILRA2, WNT9A, KRT19 and PXN.

[0019] Here, "LILRA2" refers to LILRA2 (leukocyte immunoglobulin-like receptor A2) expressed on the surface of human myeloid cells, and the LILRA2 gene is registered under Entrez Gene ID "11027". "WNT9A" refers to Wnt family member 9A expressed in organs including the heart and endometrium, and the gene is registered under Entrez Gene ID "7483". "KRT19" refers to keratin 19, a type of intermediate filament protein responsible for the structural integrity of epithelial cells, and the gene is registered under Entrez Gene ID "3880". "PXN" refers to paxillin, a cytoskeletal protein that contributes to cell adhesion to the extracellular matrix, and the gene is registered under Entrez Gene ID "5829". "PTK7" refers to protein tyrosine kinase 7, one of the transmembrane protein tyrosine kinases, and the gene is registered under Entrez Gene ID "5754". "RASA1" refers to RAS p21 protein activator 1, a member of the GAP1 family of GTPase-activating proteins localized in the cytoplasm, and the gene is registered under Entrez Gene ID "5921". "NEFL" refers to the neurofilament light chain, and the gene is Entrez Gene ID " It is registered as "4747". "IL16" refers to interleukin 16, a multifaceted cytokine that functions as a chemoattractant, a T-cell activation modulator, and an inhibitor of HIV replication. The gene is registered as Entrez Gene ID "3603". "CCN5" refers to cellular communication network factor 5, one of the members of the WNT1-induced signaling pathway, and the gene is registered with Entrez Gene ID "8839". "FMNL1" refers to a gene (formin like 1) involved in morphogenesis, cytokine disorders, and cell polarity, and the gene is registered as Entrez Gene ID "752". "SCGN" refers to secretagogin (EF-hand calcium binding protein), a calcium-binding protein in the cytoplasm, and the gene is registered with Entrez Gene ID "10590". "CXCL14" refers to a cytokine (CXC motif chemokine ligand 14) involved in immunomodulation and inflammation, and the gene is registered with Entrez Gene ID "9547". "BACH1" refers to a transcription factor (BTB domain and CNC homolog 1), and the gene is registered as Entrez Gene ID "571". "LY6D" refers to lymphocyte antigen 6 family member D, which is expressed in the extracellular region that is presumed to be involved in lymphocyte differentiation, and the gene is registered as Entrez Gene ID "8581". "CCL24" refers to CC motif chemokine ligand 24, one of the cytokines involved in immunomodulation and inflammatory processes, and the gene is registered as Entrez Gene ID "6369". "SPINT2" refers to a transmembrane protein that has inhibitory activity against various serine proteases (serine peptidase inhibitor, Kunitz type 2), and the gene is registered as Entrez Gene ID "10653". "CDH15" refers to cadherin 15, one of the calcium-dependent intercellular adhesion glycoproteins, and the gene is registered with Entrez Gene ID "1013". "CTSC" refers to the gene (cathepsin C) that encodes the peptidase C1 family and lysosomal cysteine ​​proteinase, and the gene is registered as Entrez Gene ID "1075". "CCL13" refers to CC motif chemokine ligand 13, one of the cytokines involved in immunomodulation and inflammatory processes, and the gene is registered as Entrez Gene ID "6357". "NOS1" refers to the enzyme that synthesizes nitric oxide from L-arginine (nitric oxide synthase 1), and the gene is registered as Entrez Gene ID "4842". "SCARA5" refers to a gene suspected to possess ferritin receptor activity (scavenger receptor class A member 5), and the gene is registered with Entrez Gene ID "286133". "CCL11" refers to CC motif chemokine ligand 11, one of the cytokines involved in immunomodulation and inflammatory processes, and the gene is registered as Entrez Gene ID "6356". "ACVRL1" refers to one of the type 1 cell surface receptors (activin A receptor like type 1) for the TGFβ ligand superfamily, and the gene is registered as Entrez Gene ID "94". "CD276" refers to the CD276 molecule, which is involved in regulating the T cell-mediated immune response, and the gene is registered with Entrez Gene ID "80381". "SUSD2" refers to a sushi domain containing 2 that negatively regulates the cell cycle and cell division, and the gene is registered as Entrez Gene ID "56241". "TMSB10" refers to thymosin beta 10, which is thought to be involved in the transport of actin monomers, and the gene is registered as Entrez Gene ID "9168". "NT5C3A" refers to a type of 5'-nucleotidase (cytosolic IIIA), and the gene is registered with Entrez Gene ID "51251". "TNFRSF6B" refers to a TNF receptor superfamily member 6b involved in regulating FasL and LIGHT-mediated cell death, and the gene is registered as Entrez Gene ID "8771". "SPINK1" refers to a trypsin inhibitor (serine peptidase inhibitor Kazal type 1) secreted into pancreatic juice from pancreatic acinar cells, and the gene is registered as Entrez Gene ID "6690".

[0020] In this invention, the target protein also includes proteins having an amino acid sequence substantially identical to the amino acid sequence constituting the target protein, insofar as it can serve as a biomarker for detecting diabetic neuropathy. Here, substantially identical amino acid sequences mean, for example, that when searching using the homology calculation algorithm NCBI BLAST with the conditions expected value = 10; gap allowed; filtering = ON; match score = 1; mismatch score = -3, the amino acid sequence has 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more identity with the amino acid sequence constituting the target protein.

[0021] The present invention provides a method for detecting diabetic neuropathy, which includes the step of measuring the expression level of a target protein in a biological sample taken from a subject.

[0022] In the present invention, the subjects from whom biological samples are collected are not particularly limited in terms of gender, race, etc., but diabetic patients who require detection of neuropathy or diabetic patients suspected of developing neuropathy are preferred.

[0023] The biological samples used in the present invention may be any tissues and biomaterials in which the expression of the target protein of the present invention changes in response to diabetic neuropathy. Specifically, examples include organs, skin, blood, urine, saliva, sweat, surface lipids (SSL), tissue exudates and other bodily fluids, serum prepared from blood, plasma, etc. Preferably, blood, or serum or plasma prepared from blood.

[0024] In the present invention, the target of measurement for the expression level of a target protein includes not only the protein itself, but also the RNA encoding the protein, cDNA artificially synthesized from that RNA, and genes such as the DNA encoding that RNA. Therefore, in the present invention, the expression level of a target protein comprehensively refers to the amount and activity of the target protein, as well as the expression level of the gene encoding that protein.

[0025] When measuring protein quantity as an expression level, methods such as protein chip analysis, immunoassays (e.g., various enzyme immunoassays, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), dual monoclonal antibody sandwich immunoassays, monoclonal-polyclonal antibody sandwich assays, immunostaining, immunofluorescence, Western blotting, biotin-avidin assay, immunoprecipitation, gold colloid agglutination, immunochromatography, latex agglutination (LA), immunoturbidimetry (TIA), etc.), PEA (Proximity Extension Assay), and mass spectrometry (e.g., LC-MS / MS, MALDI-TOF / MS) can be used, and the appropriate method can be selected depending on the target. For example, this can be performed by contacting a biological sample with a molecule that binds to the target protein, such as an antibody, interacting protein, ligand, nanoparticle, or aptamer, and then detecting the target protein in the sample that has bound to the molecule and measuring its level.

[0026] For example, in the Western blotting method, an antibody against the target protein is used as the primary antibody, and then an antibody that binds to the primary antibody labeled with a radioisotope, fluorescent substance, or enzyme is used as the secondary antibody to label the primary antibody. The signal derived from these labeling substances is then measured using a radiation detector, fluorescence detector, etc. Furthermore, the antibodies against the target protein mentioned above may be polyclonal or monoclonal antibodies. These antibodies can be manufactured according to known methods. Specifically, polyclonal antibodies can be obtained by using proteins expressed and purified in E. coli or other bacteria according to conventional methods, or by synthesizing a partial polypeptide of the protein according to conventional methods, immunizing non-human animals such as rabbits, and then obtaining the antibodies from the serum of the immunized animals according to conventional methods. On the other hand, monoclonal antibodies can be obtained from hybridoma cells prepared by immunizing non-human animals such as mice with proteins expressed and purified in E. coli or other bacteria according to conventional methods, or with partial polypeptides of said proteins, and then fusing the resulting spleen cells with myeloma cells. Monoclonal antibodies may also be produced using phage display (Griffiths, AD; Duncan, AR, Current Opinion in Biotechnology, Volume 9, Number 1, February 1998, pp. 102-108(7)).

[0027] When measuring the expression level of a gene (RNA, cDNA, or DNA), one can choose from nucleic acid amplification methods such as PCR, real-time RT-PCR, multiplex PCR, SmartAmp, and LAMP, which use DNA that hybridizes to these as primers; hybridization methods (DNA chips, DNA microarrays, dot blot hybridization, slot blot hybridization, Northern blot hybridization, etc.) which use nucleic acids that hybridize to these as probes; methods for determining the base sequence (sequencing); or methods combining these.

[0028] In PCR, only the DNA encoding the target protein may be amplified using a primer pair that targets the DNA encoding the target protein, or multiple DNA molecules, including the target protein, may be amplified simultaneously using multiple primer pairs. RT-PCR is an example of a method for amplifying only the DNA to be analyzed, while multiplex PCR is an example of a method for simultaneously amplifying multiple DNA molecules. Multiplex PCR is a method for simultaneously amplifying multiple gene regions by using multiple primer pairs in the PCR reaction system. Multiplex PCR can be performed using commercially available kits (e.g., Ion AmpliSeqTranscriptome Human Gene Expression Kit; Life Technologies Japan Co., Ltd., etc.).

[0029] The purification of the reaction product obtained by the PCR is preferably carried out by size separation of the reaction product. Size separation allows the target PCR reaction product to be separated from primers and other impurities contained in the PCR reaction solution. The purified PCR reaction product may be subjected to further processing necessary for subsequent quantitative analysis. For example, the purified PCR reaction product may be prepared into a suitable buffer solution for DNA sequencing, the PCR primer regions contained in the PCR-amplified DNA may be cleaved, or adapter sequences may be further added to the amplified DNA. For instance, the purified PCR reaction product can be prepared into a buffer solution, the amplified DNA can be subjected to removal of PCR primer sequences and adapter ligation, and the resulting reaction product can be amplified as needed to prepare a library for quantitative analysis.

[0030] When measuring the expression level of a gene encoding a target protein or nucleic acid derived therefrom using Northern blot hybridization, for example, a probe DNA is first labeled with a radioisotope, a fluorescent substance, etc. Then, the resulting labeled DNA is hybridized with RNA derived from a biological sample transferred to a nylon membrane, etc., according to a conventional method. Subsequently, the double helix formed between the labeled DNA and RNA is measured by detecting the signal originating from the label.

[0031] When measuring the expression level of a gene encoding a target protein or nucleic acid derived therefrom using RT-PCR, for example, cDNA is first prepared from RNA derived from a biological sample according to a standard method, and a pair of primers (a positive strand that binds to the cDNA (- strand), and a reverse strand that binds to the + strand) prepared to amplify the gene encoding the target protein are hybridized with it. Then, PCR is performed according to a standard method, and the resulting amplified double-stranded DNA is detected. For the detection of the amplified double-stranded DNA, methods such as detecting labeled double-stranded DNA produced by performing the above PCR using primers that have been previously labeled with an RI, fluorescent substance, etc., can be used.

[0032] When measuring the expression level of a gene encoding a target protein or nucleic acid derived therefrom using a DNA microarray, for example, an array in which at least one nucleic acid (cDNA or DNA) derived from the gene encoding the target protein is immobilized on a support is used. Labeled cDNA or cRNA prepared from mRNA is then bound to the microarray, and the mRNA expression level can be measured by detecting the label on the microarray. The nucleic acid immobilized on the array can be any nucleic acid that hybridizes specifically (i.e., substantially only to the target nucleic acid) under stringent conditions. For example, it may be a nucleic acid containing the entire sequence of a gene encoding a target protein, or it may be a nucleic acid consisting of a partial sequence. Here, "partial sequence" refers to a nucleic acid consisting of at least 15 to 25 bases. Here, stringent conditions can typically be washing conditions of about "1×SSC, 0.1%SDS, 37°C", more stringent hybridization conditions of about "0.5×SSC, 0.1%SDS, 42°C", and even more stringent hybridization conditions of about "0.1×SSC, 0.1%SDS, 65°C". Hybridization conditions are described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), etc.

[0033] When measuring the expression level of a gene encoding a target protein or nucleic acid derived therefrom by sequencing, one possible method is to use a next-generation sequencer (e.g., the Ion S5 / XL system, Life Technologies Japan Co., Ltd.). RNA expression can be quantified based on the number of reads generated by sequencing (read count).

[0034] Thus, the expression level of the target protein in the biological sample collected from the subject is measured, and diabetic neuropathy is detected based on this expression level. Detection is performed, for example, by comparing the measured expression level of the target protein with a control level. Here, "control level" refers, for example, to the expression level of the target protein in diabetic patients (DM) who have not developed neuropathy. The expression level in DM may be a statistical value (e.g., the mean) of the expression level of the target protein measured from the DM population. When comparing expression levels, if the expression level of the target protein derived from the subject is preferably 91% or less, more preferably 83% or less, and even more preferably 77% or less compared to the control level, the expression level of the target protein may be judged as lower than the control level. If the expression level of the target protein is preferably 110% or more, more preferably 120% or more, and even more preferably 130% or more compared to the control level, the expression level of the target protein may be judged as higher than the control level. Alternatively, the difference between the expression level of the target protein derived from the subject and the control level can be judged, for example, by whether the two are statistically significant or not. When multiple target proteins are used as target proteins, diabetic neuropathy can be detected by comparing the expression level of each target protein with a reference value and checking whether a certain percentage, for example, 50% or more, preferably 70% or more, more preferably 90% or more, and even more preferably 100%, of the expression level of the target proteins differs from the control level.

[0035] Furthermore, in this invention, diabetic neuropathy can also be detected by increasing or decreasing the expression level of the target protein. In this case, the expression level of the target protein derived from the subject is compared with the cutoff value (reference value) of the target protein. The cutoff value can be determined using various statistical analysis methods. For example, values ​​based on ROC curve (Receiver Operatorating Characteristic curve) analysis (e.g., Youden's index, distance from the upper left corner coordinate (0,1) on the ROC curve, etc.) are examples. An ROC curve is created by plotting the following parameters: the vertical axis represents the probability of a positive result in a positive patient (True Position Fraction (TPF), sensitivity), and the horizontal axis represents the value obtained by subtracting the probability of a negative result in a negative patient (Specificity) from 1 (False Position Fraction (FPF)). The ROC curve is created by varying the cutoff point, which is the threshold value for determining whether a test result indicates a finding, as a mediating variable. The cutoff point to adopt as the cutoff value from the created ROC curve should be determined based on the positioning of the test and various other conditions. Typically, if the cutoff point is set at a point with a low false positive rate, the number of negative patients who test positive will decrease, but conversely, a large number of positive patients will be excluded, resulting in lower sensitivity. On the other hand, increasing sensitivity will increase the false positive rate among negative patients. Generally, to increase both sensitivity and specificity (to approach 1), the cutoff value is set to the value that gives the point closest to (0,1) on the ROC curve, or to the value (Youden index) that maximizes "true positive (sensitivity)" - "false positive (1 - specificity)".

[0036] For example, if LILRA2, whose expression is reduced in DPN, is used as the target protein, it can be determined that a subject has diabetic neuropathy if the expression level of LILRA2 derived from that subject is lower than the cutoff value.

[0037] The present invention provides a diagnostic kit for detecting diabetic neuropathy, which contains a diagnostic reagent for measuring the expression level of a target protein in a biological sample isolated from a patient. Specifically, the kit includes reagents for immunological measurements, such as those containing molecules that bind to the target protein (e.g., antibodies that recognize the target protein), and reagents for nucleic acid amplification and hybridization, such as those containing oligonucleotides that specifically bind (hybridize) to the gene encoding the target protein or nucleic acids derived therefrom (e.g., primers for PCR). The antibodies and oligonucleotides included in the kit can be obtained by known methods as described above. Furthermore, the test kit may include, in addition to the antibodies and nucleic acids mentioned above, labeling reagents, buffer solutions, chromogenic substrates, secondary antibodies, blocking agents, equipment necessary for the test, control reagents to be used as positive and negative controls, tools for collecting samples, reagents for storing collected samples, storage containers, and reagents for extracting and purifying target proteins and nucleic acids from the samples.

[0038] Aspects and preferred embodiments of the present invention are shown below. <1> A method for detecting diabetic neuropathy in a subject, comprising the step of measuring the expression level of at least one protein selected from LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, CCN5, FMNL1, SCGN, CXCL14, BACH1, LY6D, CCL24, SPINT2, CDH15, CTSC, CCL13, NOS1, SCARA5, CCL11, ACVRL1, CD276, SUSD2, TMSB10, NT5C3A, TNFRSF6B, and SPINK1 in a biological sample taken from the subject. <2> The protein is selected from LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, SPINK1, and CCN5, preferably selected from LILRA2, WNT9A, KRT19, and PXN. <1> The method. <3> The biological sample is blood, serum, or plasma. <1> or <2> The method. <4> Measuring the expression level is equivalent to measuring the amount of the protein in question. <1> ~ <3> One of the following methods. <5> The measured expression level is compared with the reference value of the protein to assess the presence or absence of diabetic neuropathy. <1> ~ <4> One of the following methods. <6> The molecule contains a molecule that binds to the protein or an oligonucleotide that specifically hybridizes with the gene encoding the protein. <1> ~ <5> A test kit for detecting diabetic neuropathy, used in one of the following methods. <7> A marker for detecting diabetic neuropathy, comprising at least one protein selected from LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, CCN5, FMNL1, SCGN, CXCL14, BACH1, LY6D, CCL24, SPINT2, CDH15, CTSC, CCL13, NOS1, SCARA5, CCL11, ACVRL1, CD276, SUSD2, TMSB10, NT5C3A, TNFRSF6B, and SPINK1. <8> The protein is selected from LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, SPINK1, and CCN5, preferably selected from LILRA2, WNT9A, KRT19, and PXN. <7> The marker. [Examples]

[0039] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto.

[0040] (1) Method 1) Recruitment of subjects Participants were selected from 80 men and women aged 30-65 with a history of diabetes diagnosis. Selection was based on subject background information such as diabetes history, age, and sex through a web-based pre-study questionnaire. After obtaining informed consent and agreement to participate in the study, blood samples were taken and diabetic neuropathy was diagnosed. At that time, individuals who were receiving treatment or taking medication for pain or numbness, taking medication for diabetic neuropathy or analgesics, those with the following conditions (type 1 diabetes, spinal stenosis, herniated disc, osteoarthritis of the knee, rheumatoid arthritis, gout, herpes zoster, hallux valgus), those with concomitant diabetic ketoacidosis, those with serious endocrine or metabolic diseases, those with a history of diagnosis of cardiovascular diseases such as arteriosclerosis, angina pectoris, arrhythmia, or mental illness such as depression, those who had suffered severe musculoskeletal injuries such as fractures, tendon ruptures, or muscle tears in the past year, and those who were unable to walk on their own were excluded from the study.

[0041] 2) Diagnostic criteria for diabetic neuropathy (DPN) and diabetes mellitus (DM) The following examination items were performed: (1) lower limb sensation, (2) Achilles tendon reflex, and (3) vibration sensation. Patients who met two or more of the three criteria were classified as DPN. In addition, even if the above conditions were not met, patients who showed abnormal values ​​in both feet during nerve conduction studies were classified as DPN. Patients who did not meet the above conditions were classified as DM (20 DPN patients, 13 DM patients).

[0042] 2-i) Lower limb sensory abnormalities The doctor asked about the presence of pain, numbness, or decreased sensation in the lower limbs.

[0043] 2-ii) Achilles tendon reflex Participants were asked to kneel on a chair placed against the wall, facing the wall. The examiner used an Achilles tendon reflex hammer to tap the Achilles tendon and evaluated whether or not the foot plantarflexed.

[0044] 2-iii) Vibration sensation The examiner placed a vibrating tuning fork (C128) on the inner ankle of a participant who was seated. Participants were asked to report the point at which they no longer felt the vibration, and this time was recorded. Measurements were taken for both feet, and a loss of vibration sensation within 10 seconds was considered a decrease in vibration sensation.

[0045] 2-iv) Nerve conduction studies Participants lay face down on an examination table, and the area where the sensor would be applied was wiped with a special alcohol wipe (prep pad). A special gel was applied to the electrode area of ​​the nerve conduction measurement device (HDN-1000, Omron), and the electrodes were placed midway between the ankle and Achilles tendon, with the sensor part placed on the midline of the gastrocnemius muscle. A weak pulsed electric current was applied while the participant was relaxed, and nerve conduction velocity and amplitude were measured. Measurements were performed on both sides. Age and height were entered, and if the result was anything other than normal, it was judged as abnormal.

[0046] 3) Sample collection (blood sampling) Participants ate dinner by 9 PM the day before the trial and did not eat breakfast on the day of the trial before blood collection. To prevent hypoglycemia, participants were asked not to take any diabetes medication on the morning of the measurement session, but there were no restrictions on other medications. Blood was collected in a 5ml EDTA-2K vacuum blood collection tube, mixed by inverting it more than five times, and immediately cooled with ice water. Centrifuged at 1200g for 15 minutes at 4°C, and the plasma was stored at -80°C until measurement.

[0047] 4) Plasma proteome analysis For plasma proteome analysis, we used the PEA method from Olink Proteomics Co., Ltd. The PEA method is a PCR-based method for quantifying proteins by replacing the quantitative information of proteins with base sequences corresponding to those proteins. This method allows for the quantification of many types of proteins from minute sample volumes. In this study, we used an inflammatory panel (368 types) and a neurology panel (367 types), quantifying a total of 732 types of proteins (three components overlapped between the two panels).

[0048] (1) Results 1) Comparison of plasma proteomes in DPN and DM Figure 1 shows box plots of the blood concentrations of 29 markers that showed a significant difference in blood concentration between the DPN and DM groups. A t-test was used for statistical analysis, with a significance level of 0.05.

[0049] 2) Passive Action Characteristic Curves (ROCs) of Diabetic Neuropathy Using 29 Blood Markers Using relative value data for each blood marker, we attempted to perform binary classification to determine the presence or absence of diabetic neuropathy. Receiver operating characteristic curves (ROCs) were used to evaluate the classification, and the area under the ROC curve (AUC) was used to assess the model's accuracy. The results showed good model accuracy for 29 candidate markers (Table 1 below) (Figure 2, AUC ≥ 0.7).

[0050] [Table 1]

Claims

1. A method for detecting diabetic neuropathy in a subject, comprising the step of measuring the expression level of the protein LILRA2 in a biological sample taken from the subject.

2. The method according to claim 1, further comprising the step of measuring the expression level of at least one protein selected from WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, CCN5, FMNL1, SCGN, CXCL14, BACH1, LY6D, CCL24, SPINT2, CDH15, CTSC, CCL13, NOS1, SCARA5, CCL11, ACVRL1, CD276, SUSD2, TMSB10, NT5C3A, TNFRSF6B, and SPINK1.

3. The method according to claim 1, further comprising the step of measuring the expression level of at least one protein selected from WNT9A, KRT19, and PXN.

4. The method according to any one of claims 1 to 3, wherein the biological sample is blood, serum, or plasma.

5. The method according to claim 1, wherein the measurement of the expression level is the measurement of the protein amount of the protein.

6. The method according to claim 1, comprising comparing the measured expression level with a reference value of the protein to evaluate the presence or absence of diabetic neuropathy.

7. A test kit for detecting diabetic neuropathy used in the method according to claim 1, comprising a molecule that binds to the protein or an oligonucleotide that specifically hybridizes with the gene encoding the protein.

8. A marker for detecting diabetic neuropathy, consisting of LILRA2.

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