Detection method for 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 technology]

[0002] There are currently about 400 million diabetic patients, and it is said that about 12% of global health expenditures are due to diabetes or its complications. It is said that the most frightening aspect of diabetes is the complications, one of the main ones being diabetic neuropathy. Diabetic neuropathy is a nerve length-dependent neuropathy that progresses symmetrically from the extremities, initially presenting with pain and numbness, but gradually leading to decreased sensation and impaired blood flow, and in the worst cases, gangrene can lead to limb amputation, significantly reducing the patient's quality of life.

[0003] It is known that the more diabetic neuropathy progresses, the more likely it is that foot lesions, ischemic cerebrovascular disease, and ischemic heart disease will occur. It is also known that while advanced cases are difficult to cure, early cases can be improved. For these reasons, early detection and intervention of diabetic neuropathy are important.

[0004] On the other hand, there are problems with diabetic neuropathy diagnosis. That is, a nerve conduction test is necessary to make a definitive diagnosis of diabetic neuropathy, but there are problems such as the need for a dedicated machine, the need for skill in operation, time depending on the number of target nerves, and the need for electrical stimulation (pain / distress) (Non-Patent Documents 1, 2). In addition, half of diabetic neuropathy patients do not have subjective symptoms, so by the time they notice, gangrene has progressed and it is not rare for them to have to have their legs amputated.

[0005] For these reasons, there is a need to establish and popularize simple and reliable nerve monitoring techniques; however, to date no biomarkers capable of detecting diabetic neuropathy have been found. [Prior art documents] [Non-patent literature]

[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 Summary of the Invention [Problem to be solved by the invention]

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

[0008] The present inventors have discovered that there are proteins whose expression levels in blood collected from diabetic patients with and without neuropathy differ significantly, and that this can be used as an indicator to detect diabetic neuropathy.

[0009] That is, the present invention relates to the following 1) to 3). 1) A method for detecting diabetic neuropathy in a subject, comprising a 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 collected from the subject. 2) A test kit for detecting diabetic neuropathy used in the method of 1) above, which contains a molecule that binds to the protein or an oligonucleotide that specifically hybridizes to a gene that encodes the protein. 3) A marker for detecting diabetic neuropathy, which consists 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. Effect of the Invention

[0010] According to the present invention, it is possible to detect diabetic neuropathy simply and non-invasively with high accuracy, sensitivity and specificity. [Brief description of the drawings]

[0011] [Figure 1] Comparison of plasma proteomes between diabetic patients with neuropathy (DPN) and diabetic patients without neuropathy (DM). [Diagram 2] Passive operating characteristic curves (ROC) for diabetic neuropathy for 29 blood proteins. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0013] In the present invention, the term "nucleic acid" or "polynucleotide" refers to 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 the present invention, the term "gene" refers to double-stranded DNA including human genomic DNA, single-stranded DNA (positive strand) including cDNA, single-stranded DNA (complementary strand) having a sequence complementary to the positive strand, and fragments thereof, and refers to DNA that contains some biological information in the sequence information of the bases that make up the DNA. Furthermore, the "gene" in question includes not only "genes" represented by a specific base sequence, but also nucleic acids that code for their homologues (i.e., homologs or orthologs), mutants such as gene polymorphisms, and derivatives.

[0015] In the present invention, the term "diabetic neuropathy" refers to neuropathy that appears after the onset of diabetes. "Diabetic neuropathy" is divided into distal symmetric polyneuropathy and focal mononeuropathy, and the diabetic neuropathy of the present invention includes both of them. Distal symmetric polyneuropathy is divided into sensory / motor neuropathy and autonomic neuropathy. In sensory / motor neuropathy, a decrease in nerve conduction velocity is observed in nerve conduction tests from the early stage of onset, and sensory abnormalities such as spontaneous pain, numbness, paresthesia, and hypoesthesia appear in the distal lower limbs, as well as motor abnormalities such as muscle weakness, muscle atrophy, and decreased balance function. As the symptoms ascend, symptoms also appear in the distal upper limbs. In addition, eye and facial movements are also impaired. Autonomic neuropathy presents a variety of pathological conditions, including pupillary dysfunction, orthostatic hypotension, cardiac nerve disorders (sudden death, painless myocardial infarction), sweating abnormalities, gastrointestinal motility disorders (constipation, diarrhea), bladder dysfunction, and erectile dysfunction. Focal mononeuropathy includes cranial neuropathy (especially external ophthalmoplegia), neuropathy of the trunk and limbs, and diabetic amyotrophy (lumbosacral root plexus neuropathy).

[0016] The diagnosis of diabetic neuropathy is proposed to be made when two or more of the following conditions are met: 1) abnormal sensation in the lower limbs (pain, numbness, hypoesthesia), 2) decreased or absent Achilles tendon reflex on both sides, and 3) decreased vibration sense in both medial malleolus (vibration sensation of C128 tuning fork disappears within 10 seconds), or when abnormal values ​​are shown in nerve conduction tests in both feet even if these conditions are not met (Diabetic Neuropathy Society, Simplified Diagnostic Criteria for Diabetic Polyneuropathy (Mini-Revised Edition). Peripheral Nerve 23:109-111, 2012).

[0017] In the present invention, "detection of diabetic neuropathy" means clarifying the presence (with neuropathy) or absence (without neuropathy) of neuropathy in a diabetic patient. In the present invention, the term "detection" can also be replaced with the terms "examination," "measurement," "determination," "evaluation," or "evaluation assistance." In the present specification, the terms "determination" and "evaluation" do not include determination or evaluation by a doctor.

[0018] As shown in the examples below, 43 men and women with diabetes were diagnosed with or without 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 for each group, and a total of 732 proteins from the inflammatory panel (368 types) and neurology panel (367 types) were quantified (because three molecules overlapped between the two panels). As a result, the following 29 proteins were identified that showed significant differences in blood concentrations between the DPN and DM groups (Figure 1). Of these, only LILRA2 was a protein whose expression was decreased in DPN, and the others were proteins whose expression was 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 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 as Entrez Gene ID "11027." "WNT9A" refers to Wnt family member 9A, which is expressed in organs including the heart and endometrium, and the gene is registered as Entrez Gene ID "7483." "KRT19" refers to a type of intermediate filament protein (keratin 19) that is responsible for the structural integrity of epithelial cells, and the gene is registered as 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 one of the membrane-spanning protein tyrosine kinases (protein tyrosine kinase 7), and the gene is registered under the Entrez Gene ID "5754." "RASA1" refers to a member of the GAP1 family of cytoplasmic GTPase-activating proteins (RAS p21 protein activator 1), and the gene is registered under the Entrez Gene ID "5921." "NEFL" refers to the neurofilament light chain. The gene is identified by the Entrez Gene ID " It is registered as "4747". "IL16" refers to a pleiotropic cytokine (interleukin 16) that functions as a chemoattractant, a modulator of T cell activation, and an inhibitor of HIV replication, and the gene is registered as Entrez Gene ID "3603." "CCN5" refers to cellular communication network factor 5, one of the members of the WNT1-inducible signaling pathway, and the gene is registered under Entrez Gene ID "8839." "FMNL1" refers to formin like 1, which is involved in morphogenesis, cytokinesis and cell polarity, and the gene is registered under Entrez Gene ID "752." "SCGN" refers to a cytoplasmic calcium-binding protein (secretagogin, EF-hand calcium binding protein), and the gene is registered under Entrez Gene ID "10590." "CXCL14" refers to a cytokine (CXC motif chemokine ligand 14) involved in immune regulation and inflammation, and the gene is registered under 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 domain and is thought to be involved in lymphocyte differentiation. The gene is registered under the Entrez Gene ID "8581." "CCL24" refers to a cytokine (CC motif chemokine ligand 24) involved in immune regulation and inflammatory processes, and the gene is registered under Entrez Gene ID "6369." "SPINT2" is a transmembrane protein that has inhibitory activity against various serine proteases (serine peptidase inhibitor, Kunitz type 2), and the gene is registered under Entrez Gene ID "10653." "CDH15" refers to a calcium-dependent cell-cell adhesion glycoprotein (cadherin 15), and the gene is registered under Entrez Gene ID "1013." "CTSC" refers to a gene encoding a peptidase C1 family and lysosomal cysteine ​​proteinase (cathepsin C), and the gene is registered under Entrez Gene ID "1075." "CCL13" refers to a cytokine (CC motif chemokine ligand 13) involved in immune regulation and inflammatory processes, and the gene is registered under Entrez Gene ID "6357." "NOS1" refers to nitric oxide synthase 1, an enzyme that synthesizes nitric oxide from L-arginine, and the gene is registered as Entrez Gene ID "4842." "SCARA5" refers to a gene that is suspected to have ferritin receptor activity (scavenger receptor class A member 5), and the gene is registered under Entrez Gene ID "286133." "CCL11" refers to a cytokine (CC motif chemokine ligand 11) involved in immune regulation and inflammatory processes, and the gene is registered under Entrez Gene ID "6356." "ACVRL1" refers to one of the type 1 cell surface receptors for the TGFβ ligand superfamily (activin A receptor like type 1), and the gene is registered as Entrez Gene ID "94". "CD276" refers to a (CD276 molecule) involved in regulating T cell-mediated immune responses, and the gene is registered as Entrez Gene ID "80381." "SUSD2" refers to (sushi domain containing 2), which negatively regulates the cell cycle and cell division, and the gene is registered under 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 under Entrez Gene ID "9168." "NT5C3A" refers to one of the 5'-nucleotidases (5'-nucleotidase, cytosolic IIIA), and the gene is registered as Entrez Gene ID "51251." "TNFRSF6B" refers to (TNF receptor superfamily member 6b) involved in the regulation of 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 the present invention, the target protein includes a protein having an amino acid sequence substantially identical to the amino acid sequence constituting the protein, as long as it can be a biomarker for detecting diabetic neuropathy. Here, the substantially identical amino acid sequence means, for example, that when searched using the homology calculation algorithm NCBI BLAST under the conditions of expectation value=10; gaps allowed; filtering=ON; match score=1; mismatch score=-3, the amino acid sequence has an identity of 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more with the amino acid sequence constituting the protein.

[0021] The method for detecting diabetic neuropathy of the present invention comprises the step of measuring the expression level of a target protein in a biological sample collected from a subject.

[0022] In the present invention, the subject from which the biological sample is collected is not particularly limited in terms of sex or race, but is preferably a diabetic patient who requires detection of neuropathy or a diabetic patient suspected of developing neuropathy.

[0023] The biological sample used in the present invention may be any tissue or biological material in which the expression of the target protein of the present invention changes in response to diabetic neuropathy, specifically, body fluids such as organs, skin, blood, urine, saliva, sweat, surface skin lipids (SSL), tissue exudates, serum and plasma prepared from blood, and preferably blood, or serum or plasma prepared from blood.

[0024] In the present invention, the subject of measurement of the expression level of a target protein includes, in addition to the protein, an RNA encoding the protein, a cDNA artificially synthesized from the RNA, a gene encoding the RNA, a DNA, etc. Thus, in the present invention, the expression level of a target protein comprehensively means the protein amount and activity of the target protein, and the expression amount of the gene encoding the protein.

[0025] When measuring the amount of protein as the expression level, methods such as protein chip analysis, immunoassays (e.g., various enzyme immunoassays, radioimmunoassays (RIA), enzyme-linked immunoassays (ELISA), double monoclonal antibody sandwich immunoassays, monoclonal polyclonal antibody sandwich assays, immunostaining, immunofluorescence, Western blotting, biotin-avidin, immunoprecipitation, colloidal gold agglutination, immunochromatography, latex agglutination (LA), and turbidimetric immunoassay (TIA)), PEA (Proximity Extension Assay), and mass spectrometry (e.g., LC-MS / MS, MALDI-TOF / MS) can be used and can be appropriately selected depending on the subject. For example, this is carried out by contacting a molecule that binds to a target protein, such as an antibody against the target protein, an interacting protein, a ligand, a nanoparticle, an aptamer, etc., with a biological sample, detecting the target protein in the sample that is bound to the molecule, and measuring the level thereof.

[0026] For example, in the Western blot method, an antibody against a target protein is used as a primary antibody, and then an antibody that binds to the primary antibody labeled with a radioisotope, a fluorescent substance, an enzyme, or the like is used as a secondary antibody to label the primary antibody, and signals derived from these labeling substances are measured using a radiation measuring device, a fluorescence detector, or the like. The antibody against the target protein may be a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to known methods. Specifically, polyclonal antibodies can be obtained by immunizing a non-human animal such as a rabbit with a protein expressed in E. coli or the like and purified according to a conventional method, or by synthesizing a partial polypeptide of the protein according to a conventional method, and then obtaining the polyclonal antibody from the serum of the immunized animal according to a conventional method. On the other hand, monoclonal antibodies can be obtained from hybridoma cells prepared by immunizing a non-human animal such as a mouse with a protein that has been expressed and purified in Escherichia coli or a partial polypeptide of the protein according to a conventional method, and fusing the resulting spleen cells with myeloma cells. Monoclonal antibodies may also be prepared 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 amount of a gene (RNA, cDNA or DNA) as the expression level, the method can be selected from a PCR method using DNA that hybridizes to the gene as a primer, a nucleic acid amplification method represented by real-time RT-PCR, multiplex PCR, SmartAmp, LAMP, etc., a hybridization method using a nucleic acid that hybridizes to the gene as a probe (DNA chip, DNA microarray, dot blot hybridization, slot blot hybridization, Northern blot hybridization, etc.), a method for determining the base sequence (sequencing), or a combination of these methods.

[0028] In PCR, only DNA encoding the target protein may be amplified using a primer pair targeted to the DNA encoding the target protein to be analyzed, or multiple primer pairs may be used to simultaneously amplify multiple DNAs including the DNA encoding the target protein. RT-PCR is an example of a method for amplifying only the DNA to be analyzed, and multiple DNAs are an example of a method for simultaneously amplifying multiple DNAs, such as multiplex PCR. Multiplex PCR is a method for simultaneously amplifying multiple gene regions by simultaneously using multiple primer pairs in a PCR reaction system. Multiplex PCR can be performed using a commercially available kit (e.g., Ion AmpliSeq Transcriptome Human Gene Expression Kit; Life Technologies Japan, Inc., etc.).

[0029] The purification of the reaction products obtained by the PCR is preferably carried out by size separation of the reaction products, which allows the target PCR reaction products 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 required for subsequent quantitative analysis. For example, for DNA sequencing, the purified PCR reaction product may be prepared in an appropriate buffer solution, the PCR primer region contained in the PCR-amplified DNA may be cut, or an adapter sequence may be further added to the amplified DNA. For example, the purified PCR reaction product may be prepared in a buffer solution, the PCR primer sequence may be removed from the amplified DNA, and adapter ligation may be performed, and the resulting reaction product may be amplified as necessary to prepare a library for quantitative analysis.

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

[0031] When measuring the expression level of a gene encoding a target protein or a nucleic acid derived therefrom using RT-PCR, for example, first, cDNA is prepared from RNA derived from a biological sample in a conventional manner, and then a pair of primers (a positive strand that binds to the above-mentioned cDNA (-strand) and a reverse strand that binds to the + strand) that have been prepared so that the gene encoding the target protein can be amplified using this as a template are hybridized with this. Then, PCR is performed in a conventional manner, and the resulting amplified double-stranded DNA is detected. The amplified double-stranded DNA can be detected by a method of detecting labeled double-stranded DNA produced by performing the above-mentioned PCR using primers that have been labeled in advance with RI, a fluorescent substance, or the like.

[0032] When measuring the expression level of a gene encoding a target protein or a nucleic acid derived therefrom using a DNA microarray, for example, an array having at least one type of nucleic acid (cDNA or DNA) derived from a gene encoding a target protein immobilized on a support is used, labeled cDNA or cRNA prepared from mRNA is bound to the microarray, and the label on the microarray is detected, thereby measuring the expression level of mRNA. The nucleic acid immobilized on the array may 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 having the entire sequence of a gene encoding a target protein, or a nucleic acid consisting of a partial sequence. Here, the "partial sequence" may be a nucleic acid consisting of at least 15 to 25 bases. Here, the stringent conditions may be washing conditions of about "1xSSC, 0.1% SDS, 37°C", more stringent hybridization conditions may be about "0.5xSSC, 0.1% SDS, 42°C", and even more stringent hybridization conditions may be about "0.1xSSC, 0.1% SDS, 65°C". Hybridization conditions are described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press (2001), etc.

[0033] When measuring the expression level of a gene encoding a target protein or a nucleic acid derived therefrom by sequencing, for example, analysis can be performed using a next-generation sequencer (e.g., Ion S5 / XL system, Life Technologies Japan, Inc.). RNA expression can be quantified based on the number of reads (read count) generated by sequencing.

[0034] Thus, the expression level of the target protein in a biological sample collected from a subject is measured, and diabetic neuropathy is detected based on the expression level. Detection is accomplished, for example, by comparing the measured expression level of the target protein with a control level. Here, the "control level" refers to, for example, the expression level of the target protein in a diabetic patient (DM) who does not develop neuropathy. The expression level of DM may be a statistical value (e.g., an average value, etc.) of the expression level of the target protein measured from a DM population. In comparing the expression levels, if the expression level of a target protein derived from a subject is preferably 91% or less, more preferably 83% or less, and even more preferably 77% or less of the control level, the expression level of the target protein can be judged to be lower than the control level, and if the expression level of a target protein is preferably 110% or more, more preferably 120% or more, and even more preferably 130% or more of the control level, the expression level of the target protein can be judged to be higher than the control level. Alternatively, the difference between the expression level of a target protein derived from a subject and the control level can be judged, for example, by whether or not the two are statistically significantly different. 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 standard value and examining whether the expression level of a certain percentage of the target proteins, for example, 50% or more, preferably 70% or more, more preferably 90% or more, and even more preferably 100%, is different from the control level.

[0035] In addition, in the present invention, diabetic neuropathy can also be detected by an increase / decrease in the expression level of a target protein. In this case, the expression level of the target protein derived from a subject is compared with a cutoff value (reference value) of the target protein. The cutoff value can be determined by various statistical analysis methods, such as a value based on a receiver operating characteristic curve (ROC curve) analysis (e.g., Youden's index, the distance value from the upper left corner coordinate (0, 1) of the ROC curve, etc.). The ROC curve is created by plotting the probability of a positive result in a positive patient (true positive rate (TPF: True Position Fraction, sensitivity)) on the vertical axis and the value obtained by subtracting the probability of a negative result in a negative patient (specificity) from 1 (false positive rate (FPF: False Position Fraction)) on the horizontal axis, while varying the threshold for which test results are judged to indicate a finding, i.e., the cutoff point, as a parameter. The cutoff point to be used from the ROC curve can be determined based on the position of the test and other various conditions. Normally, 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, many positive patients will be excluded, resulting in low sensitivity. Conversely, if the sensitivity is increased, the false positive rate in negative patients will increase. In general, in order to increase both sensitivity and specificity (toward 1), the cutoff value is set to the value that gives the point closest to the point (0,1) on the ROC curve or the value (Youden index) at which "true positive (sensitivity)" - "false positive (1 - specificity)" is maximized.

[0036] For example, when LILRA2, the expression of which is decreased in DPN, is used as the target protein, if the expression level of LILRA2 derived from a subject is lower than a cutoff value, the subject can be determined to have diabetic neuropathy.

[0037] The test kit for detecting diabetic neuropathy of the present invention contains a test reagent for measuring the expression level of a target protein in a biological sample separated from a patient. Specifically, the test kit includes a reagent for immunological measurement, etc., containing a molecule that binds to the target protein (e.g., an antibody that recognizes the target protein, etc.), and a reagent for nucleic acid amplification and hybridization, etc., containing an oligonucleotide (e.g., a primer for PCR) that specifically binds (hybridizes) to a gene encoding the target protein or a nucleic acid derived therefrom. The antibody, oligonucleotide, etc. contained in the kit can be obtained by a known method as described above. In addition to the above-mentioned antibodies and nucleic acids, the test kit may also include labeling reagents, buffer solutions, color-developing substrates, secondary antibodies, blocking agents, equipment necessary for the test, control reagents used as positive and negative controls, tools for collecting samples, reagents for storing the 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 set out below. <1> A method for detecting diabetic neuropathy in a subject, comprising a 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 collected from the subject. <2> The protein is selected from LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, SPINK1 and CCN5, preferably LILRA2, WNT9A, KRT19 and PXN; <1> How to. <3> The biological sample is blood, serum or plasma; <1> or <2> How to. <4> The measurement of the expression level is a measurement of the protein amount of the protein; <1> ~ <3> Either way. <5> comparing the measured expression level to a reference value for said protein to assess the presence or absence of diabetic neuropathy; <1> ~ <4> Either way. <6> Containing an oligonucleotide that specifically hybridizes with a molecule that binds to the protein or a gene that encodes the protein, <1> ~ <5> A test kit for detecting diabetic neuropathy, which is used in any one of the methods above. <7> A marker for detecting diabetic neuropathy consisting 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. <8> The protein is selected from LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, SPINK1 and CCN5, preferably LILRA2, WNT9A, KRT19 and PXN; <7> Marker. EXAMPLES

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

[0040] (1) Method 1) Recruitment of subjects Participants were selected from 80 men and women aged 30-65 years who had been diagnosed with diabetes. Selection was based on subject background such as diabetes history, age, and gender through a preliminary online questionnaire, and after obtaining informed consent and consent to participate in the study, blood samples were taken and diabetic neuropathy was diagnosed. At this time, subjects were excluded from the study if they were visiting a hospital or taking medication to treat pain or numbness, taking medication for diabetic neuropathy or painkillers, had any of the following diseases (type 1 diabetes, spinal stenosis, hernia, osteoarthritis of the knee, rheumatism, gout, shingles, hallux valgus), had diabetic ketoacidosis, had serious endocrine or metabolic diseases, had a history of cardiovascular-related diseases such as arteriosclerosis, angina, or arrhythmia, or mental illness such as depression, had suffered severe damage to the musculoskeletal system in the past year such as fractures, tendon ruptures, or muscle tears, or were unable to walk on their own.

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

[0042] 2-i) Paresthesia of the lower limbs The doctor asked about the presence or absence of pain, numbness, or loss of 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 tapped the Achilles tendon with a hammer to evaluate whether the foot plantar flexed.

[0044] 2-iii) Vibration sense The examiner placed a vibrating tuning fork (C128) for vibration sense testing on the inner ankle of the participant while he or she was sitting in a chair. The participant was asked to report the time when they no longer felt the vibration, and this was taken as the measurement. Measurements were taken on both feet, and if vibration sense disappeared within 10 seconds, it was determined that the participant had a decreased vibration sense.

[0045] 2-iv) Nerve conduction tests Participants were asked to lie face down on the examination table, and the area where the sensor would come into contact was wiped with a special alcohol sheet (prep pad). Special gel was applied to the electrode sites of the nerve conduction measuring device (HDN-1000, Omron), and the electrodes were placed at the center of the ankle and Achilles tendon, with the sensor part placed on the midline of the gastrocnemius muscle. With the subject relaxed, a weak pulsed current was passed through them, and the nerve conduction velocity and amplitude were measured. Measurements were taken on both the left and right sides. Age and height were entered, and any results other than normal were deemed to be abnormal.

[0046] 3) Sample collection (blood sampling) Participants had dinner by 9 p.m. the day before the study, and had not eaten breakfast on the day of the study before blood samples were taken. To prevent hypoglycemia, participants were asked to come to the hospital without taking any diabetes medication on the morning of the measurement day, but there were no restrictions on other medications. The blood was collected in a 5 ml EDTA-2K vacuum blood collection tube, mixed by inversion at least five times, and immediately cooled in ice water. It was centrifuged at 1200 g 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, the PEA method from O-Link Proteomics, Inc. was used. The PEA method is a PCR-based method for quantifying proteins by replacing quantitative information on proteins with base sequences that correspond to the sequences of the proteins, and can quantify many types of proteins from minute samples. In this study, the inflammatory panel (368 types) and neurology panel (367 types) were used to quantify a total of 732 types of proteins (three components overlap between the two panels).

[0048] (1) Results 1) Comparison of plasma proteomes between DPN and DM The blood concentrations of 29 markers that showed significant differences between the DPN and DM groups are shown in box plots (Figure 1). Statistical analysis was performed using t-tests with a significance level of 0.05.

[0049] 2) Passive operating characteristic curve (ROC) for diabetic neuropathy using 29 blood markers Using the relative value data of each blood marker, we attempted to perform a binary classification judgment for the presence or absence of diabetic neuropathy. A receiver operating characteristic curve (ROC) was used to evaluate the classification judgment, and the accuracy of the model was evaluated by the area under the ROC curve (AUC). As a result, the model had good 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 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, ACVR1L, CD276, SUSd2, TMSB10, NT5C3A, TNFRSF6B, and SPINK1 in a biological sample collected from the subject.

2. The method according to claim 1, wherein the protein is selected from LILRA2, WNT9A, KRT19, and PXN.

3. The method according to claim 1 or 2, wherein the biological sample is blood, serum, or plasma.

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

5. The method according to claim 1, wherein the measured value of the expression level is compared with a reference value of the protein to evaluate the presence or absence of diabetic neuropathy.

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

7. A detection marker for diabetic neuropathy, consisting 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, ACVR1L, CD276, SUSd2, TMSB10, NT5C3A, TNFRSF6B, and SPINK1.

8. The marker according to claim 7, wherein the protein is selected from LILRA2, WNT9A, KRT19, and PXN.