JP2024043129A - Methods for detecting diabetic neuropathy - Google Patents

Abstract

[Problem] To provide a method for detecting diabetic neuropathy using a protein in a biological sample. [Solution] 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. [Selected Figure] None

Description

The present invention relates to a method for detecting diabetic neuropathy using a biomarker.

There are currently approximately 400 million people with diabetes, and it is said that approximately 12% of the world's health expenditures are due to diabetes or its complications. It is said that the fear of diabetes lies in its complications, and one of the main complications is diabetic neuropathy. Diabetic neuropathy is a neuropathy that progresses symmetrically from the ends of the extremities depending on the length of the nerves. Initially, it presents with pain and numbness, but gradually decreases in sensation and blood flow disorder, and in the worst case, it can lead to gangrene. This can lead to limb amputation and significantly reduce QOL.

It is known that as diabetic neuropathy progresses, the frequency of occurrence of foot lesions, ischemic cerebrovascular disease, and ischemic heart disease increases as the neuropathy progresses. It is also known that while advanced cases are difficult to cure, early cases can be improved. Based on the above, early detection and intervention of diabetic neuropathy is important.

On the other hand, there are challenges in diagnosing diabetic neuropathy. Specifically, a nerve conduction test is required to make a definitive diagnosis of diabetic neuropathy, but this has challenges such as the need for specialized equipment, the need for skilled operation, time-consuming depending on the number of nerves involved, and the involvement of electrical stimulation (pain/distress) (Non-Patent Documents 1, 2). In addition, half of diabetic neuropathy patients do not experience any 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.

For the above reasons, there is a need to establish and disseminate simple and reliable nerve monitoring technology, but so far no biomarkers that can detect diabetic neuropathy have been found.

Clinical Neurophysiology. 2018, 46: 71-77 Various scores for diabetic neuropathy. 2013, Diabetic Neuropathy (Nakayama Shoten); 37-48

The present invention relates to providing methods for detecting diabetic neuropathy using biomarkers.

The present inventors discovered that among diabetic patients, there are proteins whose expression levels in blood collected from patients with and without neuropathy are significantly different. It has been found that diabetic neuropathy can be detected as an indicator.

That is, the present invention relates to the following 1) to 3).
1) Regarding the biological samples collected from the subjects, Lilra2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, CCN5, CCN5, SCCL1, CXCL1, BACH14, LY6D, CCL24, SPINT24, SPINT24 CDH15, CTSC, CCL13, NOS1 , SCARA5, CCL11, ACVRL1, CD276, SUSD2, TMSB10, NT5C3A, TNFRSF6B, and SPINK1.
2) A test kit for detecting diabetic neuropathy used in the method of 1) above, which contains an oligonucleotide that specifically hybridizes with a molecule that binds to the protein or a gene encoding the protein.
3) LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, CCN5, FMNL1, SCGN, CXCL14, BACH1, LY6D, CCL24, SPINT2, CDH15, CTSC, CCL13, NOS1, SCARA5, C CL11, ACVRL1, CD276, A detection marker for diabetic neuropathy, comprising at least one protein selected from SUSD2, TMSB10, NT5C3A, TNFRSF6B and SPINK1.

According to the present invention, diabetic neuropathy can be detected simply and noninvasively with high accuracy, sensitivity, and specificity.

Comparison of plasma proteomes of diabetic patients with neuropathy (DPN) and diabetic patients without neuropathy (DM). Passive operating characteristic curves (ROC) of 29 blood proteins for diabetic neuropathy.

All patents, non-patent documents, and other publications cited herein are incorporated by reference in their entirety.

In the present invention, the term "nucleic acid" or "polynucleotide" means 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.

In the present invention, "gene" refers to double-stranded DNA including human genomic DNA, single-stranded DNA including cDNA (normal strand), and single-stranded DNA having a sequence complementary to the normal strand (complementary strand). , and fragments thereof, and includes some biological information in the sequence information of the bases that make up DNA.
In addition, the "gene" includes not only a "gene" expressed by a specific base sequence, but also nucleic acids encoding homologues (i.e., homologs or orthologs), variants such as genetic polymorphisms, and derivatives. be done.

In the present invention, "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.
Distally symmetrical polyneuropathy is divided into sensory/motor neuropathy and autonomic neuropathy. In sensory/motor neuropathy, a decrease in nerve conduction velocity is observed by nerve conduction tests from the early stage of onset, and a decrease in nerve conduction velocity is observed in the lower extremity. , sensory abnormalities such as spontaneous pain, numbness, paresthesia, and hypoesthesia appear, as well as motor function abnormalities such as muscle weakness, muscle atrophy, and decreased balance function, and as the symptoms progress, symptoms also appear in the distal upper limbs. Additionally, eye movements and facial movements are impaired. Autonomic nerve disorders include pupillary function abnormalities, orthostatic hypotension, cardiac nerve disorders (sudden death, painless myocardial infarction), abnormal sweating, gastrointestinal motility disorders (constipation, diarrhea), bladder dysfunction, erectile dysfunction, etc. It exhibits a variety of pathological conditions.
Focal mononeuropathies include cranial nerve disorders (particularly external ophthalmoplegia), trunk and extremity neuropathies, and diabetic muscle atrophy (lumbosacral radicular plexus neuropathy).

Diagnosis of diabetic neuropathy is as follows: 1) Lower extremity paresthesia (pain, numbness, hypoesthesia), 2) Decreased or absent bilateral Achilles tendon reflexes, 3) Decreased vibration sensation in both medial malleolus (vibration sensation of C128 tuning fork within 10 seconds) Criteria have been proposed to indicate that there is a neurological disorder if two or more of the following conditions apply, or if nerve conduction tests show abnormal values in both legs ( Diabetic Neuropathy Society, Simple Diagnostic Criteria for Diabetic Polyneuropathy (Small Revised Edition). Peripheral Nerve 23: 109-111, 2012).

In the present invention, "detection of diabetic neuropathy" means clarifying the presence (with neuropathy) or absence (with no neuropathy) of neuropathy in a diabetic patient. Note that in the present invention, the term "detection" can also be replaced with the terms inspection, measurement, determination, evaluation, or evaluation support. Note that in this specification, the term "determination" or "evaluation" does not include determination or evaluation by a doctor.

As shown in the examples below, 43 men and women with diabetes were diagnosed for the presence or absence of diabetic neuropathy using the above diagnostic criteria, and diabetic patients with neuropathy (DPN) and diabetic patients without neuropathy were diagnosed. Plasma proteome analysis was performed for each group, and a total of 732 types of proteins were quantified in the inflammatory panel (368 types) and neurology panel (367 types) (because 3 molecules overlapped between the two panels). . As a result, the following 29 types of proteins were identified, which showed a significant difference in blood concentration between the two groups, DPN and DM (FIG. 1). Among these, only LILRA2 was a protein whose expression decreased in DPN, and the others were proteins whose expression increased in DPN.
LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, CCN5, FMNL1, SCGN, CXCL14, BACH1, LY6D, CCL24, SPINT2, CDH15, CTSC, CCL13, NOS1, SCARA5, CCL 11, 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; It is more preferable to use one or more kinds.

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) 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 a cytoskeletal protein (paxillin) that contributes to cell adhesion to the extracellular matrix, and the gene is registered as Entrez Gene ID "5829."
"PTK7" refers to one of the cell membrane-spanning protein tyrosine kinases (protein tyrosine kinase 7), and the gene is registered as Entrez Gene ID "5754."
"RASA1" refers to RAS p21 protein activator 1, which is part of the GAP1 family of GTPase activating proteins localized in the cytoplasm, and the gene is registered as Entrez Gene ID "5921". There is.
"NEFL" refers to light chain neurofilament (neurofilament light chain), and the gene is Entrez Gene ID "
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 Entrez Gene ID "3603" is registered as.
“CCN5” refers to cellular communication network factor 5, which is one of the members constituting the WNT1-induced signal transduction pathway, and the gene is registered as Entrez Gene ID “8839”.
"FMNL1" refers to (formin like 1) involved in morphogenesis, cytokinesis and cell polarity, and the gene is registered as Entrez Gene ID "752".
"SCGN" refers to secretagogin, an EF-hand calcium binding protein in the cytoplasm, and the gene is registered as Entrez Gene ID "10590."
"CXCL14" refers to a cytokine involved in immune regulation and inflammation (CXC motif chemokine ligand 14), and the gene is registered as Entrez Gene ID "9547".
"BACH1" refers to one of the transcription factors (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) 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 one of the cytokines (CC motif chemokine ligand 24) involved in immune regulation and inflammatory processes, and the gene is registered as Entrez Gene ID "6369".
"SPINT2" is a transmembrane protein that has inhibitory activity against various serine proteases (serine peptide inhibitor, Kunitz type 2), and the gene is registered as Entrez Gene ID "10653". .
"CDH15" refers to one of the calcium-dependent intercellular adhesion glycoproteins (cadherin 15), and the gene is registered as Entrez Gene ID "1013".
“CTSC” refers to the peptidase C1 family and lysosomal cysteine proteinase (cathepsin C), and the gene is registered as Entrez Gene ID “1075”.
“CCL13” refers to one of the cytokines (CC motif chemokine ligand 13) involved in immune regulation and inflammatory processes, and the gene is registered as 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 scavenger receptor class A member 5 that is estimated to have ferritin receptor activity, and the gene is registered as Entrez Gene ID "286133."
“CCL11” refers to one of the cytokines (CC motif chemokine ligand 11) involved in immune regulation and inflammatory processes, and the gene is registered as Entrez Gene ID “6356”.
"ACVRL1" refers to activin A receptor like type 1, which is one of the type 1 cell surface receptors for the TGFβ ligand superfamily, and the gene is registered as Entrez Gene ID "94". There is.
"CD276" refers to a (CD276 molecule) involved in the regulation of T cell-mediated immune response, and the gene is registered as Entrez Gene ID "80381."
"SUSD2" refers to sushi domain containing 2, which negatively controls cell cycle and cell division, and the gene is registered as Entrez Gene ID "56241."
“TMSB10” refers to thymosin beta 10 (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 (5'-nucleotidase, cytosolic IIIA), which is one of 5'-nucleotidases, and the gene is registered as Entrez Gene ID "51251".
“TNFRSF6B” refers to TNF receptor superfamily member 6b (TNF receptor superfamily member 6b), which is 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 from pancreatic acinar cells into pancreatic juice, and the gene is registered as Entrez Gene ID "6690" .

In the present invention, the target protein includes a protein having substantially the same amino acid sequence as the amino acid sequence constituting the protein, as long as it can serve as a biomarker for detecting diabetic neuropathy. Here, a substantially identical amino acid sequence is defined as, for example, using the homology calculation algorithm NCBI BLAST and using the following conditions: Expected value = 10; Gaps allowed; Filtering = ON; Match score = 1; Mismatch score = -3. When searched for, it means that there is 90% or more identity, preferably 95% or more, more preferably 98% or more, still more preferably 99% or more identity with the amino acid sequence constituting the protein.

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

In the present invention, the subject from whom a biological sample is collected is not particularly limited in terms of gender or race, but is preferably a diabetic patient whose neuropathy needs to be detected or a diabetic patient who is suspected of developing neuropathy.

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 depending on diabetic neuropathy. Specific examples include body fluids such as organs, skin, blood, urine, saliva, sweat, skin surface lipids (SSL), tissue exudates, and serum and plasma prepared from blood, preferably blood or blood. Examples include prepared serum or plasma.

In the present invention, targets for measuring the expression level of a target protein include, in addition to the protein, genes such as RNA encoding the protein, cDNA artificially synthesized from the RNA, and DNA encoding the RNA. . Therefore, in the present invention, the expression level of a target protein comprehensively refers to the protein amount and activity of the target protein, and the expression level of the gene encoding the protein.

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 immunosorbent assays (ELISA), double monoclonal antibody sandwich immunoassays, monoclonal polyclonal antibody sandwich assays, immunostaining, immunofluorescence, Western blotting, biotin-avidin, 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 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.

For example, according to Western blotting, 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 radioactive isotope, a fluorescent substance, an enzyme, etc. is used as a secondary antibody, and the primary antibody is are labeled, and signals derived from these labeled substances are measured using a radiation meter, a fluorescence detector, etc.
Note that 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, a polyclonal antibody is produced by immunizing a non-human animal such as a domestic rabbit using a protein expressed and purified in Escherichia coli etc. according to a conventional method, or by synthesizing a partial polypeptide of the protein according to a conventional method. It can be obtained from the serum of the immunized animal according to a conventional method.
On the other hand, monoclonal antibodies are produced by immunizing a non-human animal such as a mouse with a protein expressed and purified in Escherichia coli etc. or a partial polypeptide of the protein according to a conventional method, and then fusing the obtained spleen cells with myeloma cells. It can be obtained from prepared hybridoma 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)).

When measuring the expression level of a gene (RNA, cDNA, or DNA), typical methods include PCR, real-time RT-PCR, multiplex PCR, SmartAmp, and LAMP using DNA that hybridizes to these as primers. methods for amplifying nucleic acids, hybridization methods using nucleic acids that hybridize to these as probes (DNA chips, DNA microarrays, dot blot hybridization, slot blot hybridization, Northern blot hybridization, etc.), methods for determining base sequences ( (sequencing) or a combination of these methods.

In PCR, a primer pair targeted to the DNA encoding the target protein to be analyzed may be used to amplify only the DNA encoding the target protein, 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.).

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 into a suitable 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 into 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.

When measuring the expression level of a gene encoding a target protein or a nucleic acid derived therefrom using Northern blot hybridization, for example, the probe DNA is first labeled with a radioactive isotope, a fluorescent substance, etc., and then the obtained The labeled DNA is hybridized with RNA derived from a biological sample transferred to a nylon membrane or the like according to a conventional method. Thereafter, a method of measuring the formed double strand of labeled DNA and RNA by detecting a signal derived from the labeled substance is included.

When measuring the expression level of a gene encoding a target protein or a nucleic acid derived therefrom using RT-PCR, for example, first prepare cDNA from RNA derived from a biological sample according to a conventional method, and use this as a template to inject the target protein. 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 cDNA are hybridized with this. Thereafter, PCR is performed according to a conventional method, and the resulting amplified double-stranded DNA is detected. To detect the amplified double-stranded DNA, use a method such as detecting labeled double-stranded DNA produced by performing the above PCR using primers that have been labeled in advance with RI, a fluorescent substance, etc. Can be done.

When measuring the expression level of a gene encoding a target protein or a nucleic acid derived therefrom using a DNA microarray, for example, at least one type of nucleic acid (cDNA or DNA) derived from a gene encoding the target protein is immobilized on a support. The expression level of mRNA can be measured by binding labeled cDNA or cRNA prepared from mRNA onto the microarray using the labeled array, and detecting the label on the microarray.
The nucleic acids to be immobilized on the array may be any nucleic acids that hybridize specifically (that is, substantially only to the nucleic acid of interest) under stringent conditions, such as those of genes encoding target proteins. The nucleic acid may have the entire sequence or may consist of a partial sequence. Here, the "partial sequence" includes a nucleic acid consisting of at least 15 to 25 bases. Stringent conditions here usually include washing conditions such as "1x SSC, 0.1% SDS, 37°C", and more severe hybridization conditions include "0.5x SSC, 0.1% SDS, 37°C". % SDS, 42° C., and even more severe hybridization conditions include conditions such as “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.

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 (for example, Ion S5/XL system, Life Technologies Japan Co., Ltd.). It will be done. RNA expression can be quantified based on the number of reads generated by sequencing (read count).

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 a target protein in a diabetic patient (DM) who has not developed neuropathy. The expression level of a DM may be a statistical value (e.g., an average value, etc.) of the expression level of a 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 reference 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.

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, a distance value from the upper left corner coordinates (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)), 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)) on the horizontal axis, while varying the threshold for determining which test result value indicates the presence of a finding, that is, the cutoff point, as a parameter.
The cutoff point to be adopted from the created ROC curve can be determined based on the positioning of the test and various other 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 (to approach 1), the cutoff value is set to a value that gives the closest point to point (0, 1) on the ROC curve or a value (Youden index) that maximizes "true positive (sensitivity)" - "false positive (1 - specificity)."

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

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 isolated from a patient. Specifically, reagents for immunoassays that contain molecules that bind to target proteins (e.g., antibodies that recognize target proteins), and reagents that specifically bind to genes encoding target proteins or nucleic acids derived therefrom ( Examples include reagents for nucleic acid amplification and hybridization, including oligonucleotides that hybridize (eg, primers for PCR). The antibodies, oligonucleotides, etc. included in the kit can be obtained by known methods as described above.
In addition to the antibodies and nucleic acids mentioned above, the test kit also includes labeling reagents, buffers, coloring substrates, secondary antibodies, blocking agents, equipment necessary for the test, control reagents used as positive controls and negative controls, It can include tools for collecting a sample, reagents for preserving the collected sample, containers for preservation, reagents for extracting and purifying target proteins and nucleic acids from the sample, and the like.

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 method according to <1>, wherein the protein is selected from LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, SPINK1 and CCN5, preferably selected from LILRA2, WNT9A, KRT19 and PXN.
<3> The method according to <1> or <2>, wherein the biological sample is blood, serum or plasma.
<4> The method according to any one of <1> to <3>, wherein the measurement of the expression level is the measurement of the protein amount of the protein.
<5> The method according to any one of <1> to <4>, further comprising comparing the measured expression level with a reference value of the protein to evaluate the presence or absence of diabetic neuropathy.
<6> A test kit for detecting diabetic neuropathy, which is used in any one of the methods <1> to <5>, comprising a molecule that binds to the protein or an oligonucleotide that specifically hybridizes to a gene that encodes the protein.
<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 marker according to <7>, wherein the protein is selected from LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, SPINK1 and CCN5, preferably selected from LILRA2, WNT9A, KRT19 and PXN.

Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited thereto.

(1) Method 1) Recruitment of subjects The subjects were selected from among 80 men and women aged 30 to 65 who had been diagnosed with diabetes. Subjects were selected based on background information such as history of diabetes, age, and gender through a preliminary online questionnaire.After obtaining informed consent and consent to participate in the study, blood was drawn and diagnosis of diabetic neuropathy was conducted. . At that time, those who are visiting the hospital or taking medication to cure pain or numbness, those who are taking medications for diabetic neuropathy or painkillers, and those who have the following diseases (type 1 diabetes, spinal stenosis, hernia, etc.) (knee osteoarthritis, rheumatism, gout, shingles, bunions), diabetic ketoacidosis, severe endocrine and metabolic diseases, arteriosclerosis, angina pectoris, Those who have been diagnosed with cardiovascular-related diseases such as arrhythmia or mental illnesses such as depression, those who have suffered severe injuries to their locomotor organs such as fractures, tendon ruptures, and muscle strains in the past year, and those who are unable to walk on their own. were excluded from the study subjects.

2) Diagnostic criteria for diabetic neuropathy (DPN) and diabetes mellitus (DM) The following examination items ((1) lower extremity sensation, (2) Achilles tendon reflex, and (3) vibration sensation) were examined, and 2 of the 3 items were examined. Those who met or exceeded the criteria were designated as DPN. In addition, even if the above conditions were not met, if both legs showed abnormal values in the nerve conduction test, the patient was classified as DPN. Those who did not meet the above conditions were designated as DM (20 DPNs, 13 DMs).

2-i) Lower extremity paresthesia A doctor asked patients whether they had pain, numbness, or hypoesthesia in their lower extremities.

2-ii) Achilles tendon reflex Participants were asked to take a kneeling position on a chair set up against a wall, facing the wall. The examiner used a hammer for Achilles tendon reflex testing to strike the Achilles tendon and evaluate whether the foot would plantar flex.

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/she was sitting in a chair. The participant was asked to report the time when they no longer felt the vibration, and this time was taken as the measurement value. Measurements were taken on both feet, and if the vibration sense disappeared within 10 seconds, it was judged that the vibration sense had decreased.

2-iv) Nerve conduction test 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, and the sensor part was placed on the midline of the gastrocnemius muscle. With the subject relaxed, a weak pulsed current was passed through the device, and the nerve conduction velocity and amplitude were measured. Measurements were performed on both the left and right sides. Age and height were entered, and any results other than normal were deemed to be abnormal.

3) Specimen collection (blood collection)
Blood samples were taken after participants had eaten dinner by 9:00 pm on the day before participating in the test, and had not eaten breakfast on the day of participating in the test. To prevent hypoglycemia on the morning of the measurement session, the subjects came to the hospital without taking any diabetes medication, and there were no restrictions on the use of other medications.
The blood was collected into an EDTA-2K 5ml vacuum blood collection tube, mixed by inversion five or more times, and immediately cooled with ice water. Centrifugation was performed at 1200g x 15 minutes at 4°C, and the plasma was stored at -80°C until measurement.

4) Plasma proteome analysis For plasma proteome analysis, the PEA method of O-Link Proteomics Co., Ltd. was used. The PEA method is a PCR-based method for quantifying proteins by replacing protein quantitative information with a base sequence that has a sequence corresponding to the protein, and it is possible to quantify many types of proteins from a small amount of sample. . In this test, a total of 732 types of proteins were quantified using an inflammatory panel (368 types) and a neurology panel (367 types) (3 components overlapped between the two panels).

(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.

2) Passive operating characteristic curve (ROC) of diabetic neuropathy using 29 types of blood markers
Using the relative value data of each blood marker, we attempted to perform a binary classification judgment on 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 accuracy as a model was good for 29 types of candidate markers (Table 1 below) (FIG. 2, AUC≧0.7).

Claims (8)

Regarding biological samples collected from subjects, LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, CCN5, FMNL1, SCGN, CXCL14, BACH1, LY6D, CCL24, SPINT2, CDH15, CTSC, CCL13, NOS1, S CARA5 A method for detecting diabetic neuropathy in a subject, the method comprising measuring the expression level of at least one protein selected from , CCL11, ACVRL1, CD276, SUSD2, TMSB10, NT5C3A, TNFRSF6B and SPINK1. 2. The method of 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. The method according to any one of claims 1 to 3, wherein the measurement of the expression level is the measurement of the protein amount of the protein. The method according to any one of claims 1 to 4, wherein the measured expression level is compared to a reference value for the protein to assess the presence or absence of diabetic neuropathy. A method for detecting diabetic neuropathy used in the method according to any one of claims 1 to 5, comprising an oligonucleotide that specifically hybridizes with a molecule that binds to the protein or a gene encoding the protein. test kit. LILRA2, WNT9A, KRT19, PXN, PTK7, RASA1, NEFL, IL16, CCN5, FMNL1, SCGN, CXCL14, BACH1, LY6D, CCL24, SPINT2, CDH15, CTSC, CCL13, NOS1, SCARA5, CCL 11, ACVRL1, CD276, SUSD2, A detection marker for diabetic neuropathy, comprising at least one protein selected from TMSB10, NT5C3A, TNFRSF6B and SPINK1. The marker according to claim 7, wherein the protein is selected from LILRA2, WNT9A, KRT19 and PXN.