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WO2020209469A1 - Biomarqueur pour diagnostiquer chaque sous-type de neuropathie périphérique - Google Patents

Biomarqueur pour diagnostiquer chaque sous-type de neuropathie périphérique Download PDF

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WO2020209469A1
WO2020209469A1 PCT/KR2019/016923 KR2019016923W WO2020209469A1 WO 2020209469 A1 WO2020209469 A1 WO 2020209469A1 KR 2019016923 W KR2019016923 W KR 2019016923W WO 2020209469 A1 WO2020209469 A1 WO 2020209469A1
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peripheral neuropathy
neuropathy
ncam
protein
expression level
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Korean (ko)
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박환태
김종국
김영희
한진영
최병옥
김영혜
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Sugentech Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to a biomarker capable of effectively diagnosing various peripheral neuropathies by subtype.
  • the peripheral nervous system includes the motor and sensory roots, the dorsal root ganglion, the nerve plexus, and the peripheral nerves, and the cranial nerves and ganglions excluding the optic and olfactory nerves, as well as the autonomic and autonomic nerves. Include.
  • peripheral neuropathy there are various pathological findings that invade the peripheral nerve, and when physical or chemical damage to axons occurs, Waller degeneration occurs at the distal part of the damaged area, and metabolic disorders of neurons.
  • Axonal degeneration which proceeds from the distal to the proximal part of the axon (dying-back), neuropathy that causes the degeneration of the cell body and axon at the same time, and the function of the axon is relatively maintained while myelinated ( Myelin sheath) is primarily destroyed, such as demyelinopathy, which can be mixed in certain peripheral neuropathies.
  • Symptoms of peripheral neuropathy include sensory symptoms, motor symptoms, and autonomic symptoms, and sensory symptoms and motor symptoms are divided into positive and negative symptoms.
  • Voice motor symptoms are due to the loss of a conduction block or axon of the motor nerve and appear as muscle weakness.
  • the symptoms of benign movements appear as fasciculation, myokymia, tremor, and muscle cramp due to abnormal activity in the peripheral nerve.
  • Benign sensory symptoms include hypersensitivity, including pain, and dysethesia, and negative sensory symptoms may include decreased sensitivity and numbness.
  • Autonomic nervous system disorders can be accompanied by digestive disorders or general autonomic disorders in the body.
  • peripheral neuropathy causes of peripheral neuropathy are secondary neurodegeneration following physical nerve damage such as compression or entrapment, acquired immunodeficiency syndrome, infectious diseases such as leprosy, Lyme disease, medical diseases such as diabetes, ischemic disease, There are a wide variety of cases, such as paraneoplastic syndrome, nutritional deficiencies, toxic diseases, inflammatory demyelinating diseases, and genetic diseases.
  • the causes are very diverse, and even a hospital specializing in peripheral neuropathy cannot determine the cause of about 25%. Therefore, in order to accurately diagnose peripheral neuropathy, a step-by-step and systematic approach is required through various laboratory tests including detailed medical history, physical and neurological examination, and electrophysiological examination.
  • Demyelinating peripheral neuropathy is partially differentiated from axonopathy by electrophysiological examination, but there is still no method for discriminating and diagnosing inflammatory or hereditary causes within demyelination using specific proteins in the blood, and the introduction of such a simple diagnostic marker is a demyelination disease. It is also very important in selecting treatment strategies through rapid diagnosis of subtypes.
  • the scientific background for the invention of a subtype-specific biomarker for peripheral neuropathy is due to the study of transformation of Schwann cells directly involved in demyelination.
  • Mature Schwann cells that produce myelin are transformed into immature Schwann cells during inflammatory demyelination, contributing to demyelination, and express a specific phenotypic factor.
  • hereditary demyelination it shows a phenotype that maintains the immature state during development or causes abnormal differentiation.
  • Schwann cells when axonal damage occurs are also dedifferentiated and transformation into immature Schwann cells occurs.
  • the present invention provides a method of providing information for predicting or diagnosing the risk of developing peripheral neuropathy by using a combination of biomarkers, providing a diagnostic composition for each subtype, and providing a drug screening method for treating peripheral neuropathy using this Has its purpose.
  • the hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polymyelopathy (CIDP, Chronic inflammatory neuropathy). Demyelinating polyradiculoneuropathy) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP), a method of providing information for predicting or diagnosing the risk of developing peripheral neuropathy subtypes.
  • CMT1a Charcomaritus disease 1a
  • AMAN acute motor axonal neuropathy
  • CIDP chronic inflammatory demyelinating polymyelopathy
  • Demyelinating polyradiculoneuropathy or acute inflammatory demyelinating polyradiculoneuropathy (AIDP)
  • sample is at least one selected from the group consisting of serum, plasma, nerve cells, immune cells, cerebrospinal fluid, and exosomes.
  • the peripheral neuropathy is an inherited peripheral neuropathy or a non-hereditary peripheral neuropathy
  • the hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polymyelopathy (CIDP, Chronic inflammatory neuropathy). Demyelinating polyradiculoneuropathy) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP), a method for screening candidates for prevention or treatment of peripheral neuropathy subtypes.
  • CMT1a Charcomaritus disease 1a
  • AMAN acute motor axonal neuropathy
  • CIDP Chronic inflammatory demyelinating polyradiculoneuropathy
  • AIDP acute inflammatory demyelinating polyradiculoneuropathy
  • sample is at least one selected from the group consisting of serum, plasma, nerve cells, immune cells, cerebrospinal fluid, and exosomes.
  • a nucleotide sequence of a gene encoding p75 and NCAM protein a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance that specifically binds to a protein encoded by the nucleotide sequence,
  • peripheral neuropathy For diagnosis of the onset of inherited peripheral neuropathy or non-hereditary peripheral neuropathy,
  • the hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polymyelopathy (CIDP, Chronic inflammatory neuropathy). Demyelinating polyradiculoneuropathy) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP), a composition for diagnosing peripheral neuropathy subtypes.
  • CMT1a Charcomaritus disease 1a
  • AMAN acute motor axonal neuropathy
  • CIDP chronic inflammatory demyelinating polymyelopathy
  • Demyelinating polyradiculoneuropathy Demyelinating polyradiculoneuropathy
  • AIDP acute inflammatory demyelinating polyradiculoneuropathy
  • composition of the above 13, further comprising a nucleotide sequence of a gene encoding CXCL13 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance that specifically binds to a protein encoded by the nucleotide sequence .
  • composition of 13 above, wherein the composition is for diagnosis of inflammatory demyelinating polymuscular neuropathy.
  • peripheral neuropathy For diagnosis of the onset of inherited peripheral neuropathy or non-hereditary peripheral neuropathy,
  • the hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polymyelopathy (CIDP, Chronic inflammatory neuropathy). Demyelinating polyradiculoneuropathy) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP), a diagnostic kit for peripheral neuropathy subtypes.
  • kit is a protein array or a protein chip including a substance specifically binding to a protein encoded by the nucleotide sequence.
  • the information providing method of the present invention can effectively provide information for predicting or diagnosing the risk of developing a peripheral neuropathy subtype by measuring the expression level of a biomarker.
  • the screening method of the present invention can effectively screen a candidate therapeutic drug for peripheral neuropathy by measuring the expression level of a biomarker.
  • the diagnostic composition and kit of the present invention can effectively diagnose peripheral neuropathy.
  • FIG. 1A to 1C (A) Workflow for biomarker development in demyelinating neuropathy, (B) Venn diagram depicting the overlap of the proteome in the mouse Schwann cell exosomes of this study and the ExoCarta mouse exosomes, (C ) Characteristics of Schwann cell exosomes by Western blot analysis (SCL: Schwann cell lysate, Rab5b: cytoplasmic protein marker), (D, E) detection results of p75 and NCAM in the serum of peripheral neuropathy patients by ELISA (ANOVA Significant differences between the patient group and the normal control group by (represented by **(p ⁇ 0.01) and ***(p ⁇ 0.005)), (F, G) ROC curves of p75 and NCAM detection by ELISA.
  • FIG. 2a to 2b is the expression profile of (A) p75 and (B) NCAM in the neuromuscular of human CIDP and CMT1a patients, where arrows are demyelinating Schwann cells, arrowheads are NCAMs in non-myelinated Schwann cells. , Asterisks indicate excess onion progenitor Schwann cells, double arrows indicate demyelinating Schwann cells, respectively (scale bar, 20 ⁇ m).
  • C is the expression profile of NCAM in the nerves of human CIDP and CMT1a patients, and NCAM staining of the longitudinal section (left two panels) showed differences in NCAM expression in the splenic nerves of CIDP and CMT1a (MBP: myelin basic protein, Asterisk: Schmidt-Lanterman incisures, scale bar, 20 ⁇ m).
  • MBP myelin basic protein
  • Asterisk Schmidt-Lanterman incisures, scale bar, 20 ⁇ m
  • the right panel is an enlarged image of onion progenitor cells of CMT1a (arrow: nucleus). All concentric cytoplasmic layers of onion progenitor Schwann cells expressed NCAM.
  • (D) is a graph showing the percentage of demyelinating Schwann cells expressing p75 or NCAM among 600 to 1800 MBP positive Schwann cells in the nerve sections of CIDP and CMT1a patients (Unpaired Student's t-test, *: p ⁇ 0.05, **: p ⁇ 0.01).
  • (E) is the fluorescence intensity values of p75 and NCAM staining in an in vivo nerve biopsy (Unpaired Student's t-test, **: p ⁇ 0.01).
  • 3A to 3B shows that the osteotomy induced the expression of p75, not NCAM, while demyelination of the SC (DSC, arrow), and only immature Schwann cells (arrowheads) showed NCAM expression before and after axonal dissection. (Scale bar, 20 ⁇ m).
  • B is a Western blot analysis result showing that the expression of p75, not NCAM, was induced in the sciatic nerve after axonal incision.
  • C shows the quantitative values of p75 and NCAM expression levels (mean ⁇ SEM, unpaired Student's t-test, **: p ⁇ 0.01 in three independent experiments).
  • (D) shows that demyelinating Schwann cells at the beginning and peak stages of EAN showed p75 induction (arrow), but NCAM staining increased in immature Schwann cells (arrowheads).
  • (E) is a Western blot analysis result showing that p75 and NCAM are induced in the sciatic nerve of EAN.
  • (F) is a graph showing the quantitative values of p75 and NCAM expression levels (mean unpaired Student's t-test from 3 independent experiments, *: p ⁇ 0.05, **: p ⁇ 0.01).
  • Figure 4 Western blot analysis results of analyzing the expression patterns of p75 and NCAM in primary cultured Schwann cells by cytokines and growth factors.
  • Pro-inflammatory cytokines such as INF- ⁇ and TNF- ⁇ increased NCAM expression in cultured primary SCs, but ER stress inducers, including proteasome inhibitors (Bortezomib, BTZ) and thapsigargin (TG) 45, did not.
  • the expression of p75 was not significantly changed by pro-inflammatory cytokine treatment.
  • the graph shows the quantitative value of the NCAM expression level in three independent experiments (Unpaired student t-test; p: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001).
  • 5A to 5B Regarding the proinflammatory environment associated with M1-macrophages having CXCL13 expression in B7-2KO neurons, (A) cytokine arrays in B7-2KO neurons and injured neurons. (B) Schematic of the cytokine expression profile in B7-2KO neurons and injured C57BL/6 neurons, where the levels of CXCL13, CCL5, MIP-1 and CXCL10 were specifically increased in B7-2KO neurons. (C) Expression of CXCL13 was induced in CD68+ macrophages (arrowheads), but was not expressed in S100-positive SCs in B7-2KO neurons.
  • CXCL13 positive cells were not present in the NOD nerve or the damaged C57BL/6 nerve (6dPI, 6 days after injury) (WD: Wallerian degeneration, scale bar, 20 ⁇ m).
  • D Using antibodies against CD206 and CD197, M2 and M1 macrophages were detected and analyzed in the nerve section, respectively. In B7-2KO neurons, most of the CD68+ macrophages were CD206, and some CD197+ cells were present. During WD, most of the CD68+ macrophages were CD206+ (arrowheads) and a few were CD197+ (scale bar, 20 ⁇ m).
  • FIG. 7 The serum CXCL13 concentration of patients with peripheral neuropathy was investigated using ELISA. Serum concentrations of CXCL13 were significantly increased in patients with acute (AIDP; p ⁇ 0.05) and chronic (CIDP; p ⁇ 0.01) inflammatory demyelinating polyneuromyopathy compared to healthy controls, acute motor axon neuropathy (AMAN), and CMT1a group. I did. (Significant differences between the patient group and the healthy control group by repeated measurement ANOVA are expressed as * (p ⁇ 0.05) and ** (p ⁇ 0.01)) (B, C) CXCL13 expression in neuromuscular muscles of human CIDP and CMT1a patients With arrows, CXCL13+ cells in the cranial nerves are indicated. The gastrocnemius nerve of CMT1a patient showed myelin dysplasia without significant expression of CXCL13 (scale bar, 50 ⁇ m).
  • 8A to 8D (A) Myelination profile of the sciatic nerve of wild type (WT) and C22 mice, Semitin plastic sections showed significant hypomyelination in C22 mice (5W; postnatal 5 weeks, scale bar, 20 ⁇ m), (B) NCAM expression profile of the sciatic nerve of C22 and WT mice at 8 weeks postnatal (8W), NCAM expression in adult normal mice (WT) was non-myelinated SC (arrowhead) And a number of small NCAM positive staining (arrows) were observed in C22 mice (scale bar, 20 ⁇ m).
  • Figure 9 Schematic explaining the selective expression of NCAM in inflammatory demyelinating neuropathy.
  • inflammatory demyelinating neuropathy such as acute (AIDP) and chronic (CIDP) inflammatory demyelinating neuropathy
  • the inflammatory environment of M1-type macrophages expressing CXCL13 induces SC dedifferentiation and NCAM expression.
  • SCs dedifferentiated in both conditions generally express c-Jun, p75 and CCL2 and contribute to myelination.
  • Figure 10 As a pathological immature Schwann cell model in peripheral neuropathy, a schematic showing the difference between demyelinating Schwann cells and pathologically differentiated Schwann cells in peripheral neuropathy based on p75 and NCAM expression.
  • Demyelinating SC p75+/NCAM-
  • extra SC p75-/NCAM+
  • Can be ID: inflammatory demyelination
  • the peripheral nervous system refers to the rest of the nervous system of our body central nervous system that is, the brain and spinal cord, and is distributed in almost all organs in the body and is involved in the regulation of its functions.
  • the peripheral nervous system is largely motor nervous system, sensory nervous system, autonomic It is classified as the nervous system.
  • Peripheral neuropathy is classified into congenital or hereditary peripheral neuropathy, acquired peripheral neuropathy depending on the age of occurrence and family history, and it is classified into sensory, motility, autonomic neuropathy, and complex depending on the area where the disorder mainly appears.
  • pathology There are classifications according to phenomena and classification according to the distribution of invading nerves.
  • CMT Charcot-Marie-Tooth disease
  • HNPP hereditary compression neuropathy
  • HMN Hereditary motor neuropathy
  • HSAN hereditary sensory autonomic neuropathy
  • CMT congenital or hereditary peripheral neuropathy
  • CMT Charcot-Marie-Tooth disease
  • HNPP hereditary compression neuropathy
  • HMN Hereditary neuropathy with liability to pressure palsies
  • HSAN hereditary sensory autonomic neuropathy
  • HSAN Hereditary sensory and autonomic neuropathy
  • peripheral neuropathies including congenital or hereditary peripheral neuropathy may be included.
  • Metabolic neuropathy addictive neuropathy, allergic neuropathy, cancerous neuropathy, other neuropathy It may include.
  • acquired peripheral neuropathy include Inflammatory demyelinating polyradiculoneuropathy, Acute motor axonal neuropathy (AMAN), Guillain-Barre syndrome (GBS), Miller Fisher syndrome (MFS, Miller-Fisher syndrome), diabetic peripheral neuropathy, vasculitis neuropathy, etc.
  • the inflammatory demyelinating polyradiculoneuropathy is chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).
  • Chronic inflammatory demyelinating polyradiculoneuropathy Acute inflammatory demyelinating polyradiculoneuropathy (AIDP), Acute sensory ataxic neuropathy (ASAN), Multifocal motor neuropathy (MMN) And Lewis-sumner syndrome (LSS).
  • the p75 of the present invention is a neurotrophin receptor, which contains four TNFR cysteine-rich motifs, a transmembrane region, and an intracellular region containing a death domain, and thus, LINGO-1/Nogo-66 receptor signals It has been known that it is a component of the ring pathway and can mediate the survival and death of neurons, but its role in relation to specific peripheral neuropathy is unknown.
  • NCAM Neuronal cell adhesion molecule 1 of the present invention is known as a binding glycoprotein expressed on the surface of neurons, glial cells or skeletal muscle, also called CD56, but no known about its role in relation to specific peripheral neuropathy.
  • CXCL13 (C-X-C motif chemokine ligand 13) of the present invention belongs to the CXC chemokine family, and plays an important role in lymphoid organ formation and development, B cell follicle formation, and B cell supplementation. It is ectopically produced in inflammatory tissues of multiple chronic inflammatory diseases, and is considered to play an important role in maintaining local B and T cell activity and inflammation, but no known role is known in relation to peripheral neuropathy.
  • the present invention provides a method of providing information for predicting and diagnosing the risk of development of each subtype of peripheral neuropathy, including measuring the expression level of p75 and NCAM proteins in a sample isolated from an individual.
  • the present invention finds that the expression patterns of p75 and NCAM in samples obtained from patients with peripheral neuropathy are different for each peripheral neuropathy subtype, and based on this, the combination of p75 and NCAM is a peripheral neuropathy subtype-specific biomarker or indicator It is based on discovering its applicability as
  • the individual refers to an animal including humans, and specifically, may mean at least one selected from human, rat, rabbit, mouse, etc., but is not particularly limited if it is a possible target of peripheral neuropathy.
  • the method may measure the expression level of p75 and NCAM proteins, and according to the result, the risk of developing hereditary peripheral neuropathy or non-hereditary peripheral neuropathy may be predicted or determined whether to develop.
  • the method may further include predicting that the risk of developing hereditary peripheral neuropathy is higher if there is no significant difference in the expression level of the p75 protein compared to the control individual, and the expression level of NCAM is significantly higher than that of the control individual.
  • the hereditary peripheral neuropathy may be Charcomaritus disease 1a (CMT1a).
  • the method may further include predicting that the risk of developing non-hereditary peripheral neuropathy is higher when the expression level of p75 and NCAM protein is significantly higher than that of the control individual.
  • the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), or acute inflammatory demyelinating polymuscular neuropathy. It may be a disease (AIDP, Acute inflammatory demyelinating polyradiculoneuropathy).
  • the method is CMT1a hereditary peripheral neuropathy; Or, it is possible to predict and determine the risk of onset or onset of non-hereditary peripheral neuropathy, which is AMAN, CIDP, or AIDP.
  • the method may further include the step of measuring the expression level of the CXCL13 protein.
  • inflammatory demyelinating polyradiculoneuropathy (Inflammatory demyelinating polyradiculoneuropathy) Predicting that the risk of developing is higher may be further included.
  • the inflammatory demyelinating polymuscular neuropathy may include chronic or acute inflammatory demyelinating polymuscular neuropathy.
  • the criterion is a confidence level of 95% (p ⁇ 0.05).
  • it may be a confidence level of 99% (p ⁇ 0.01), for example, by the Unpaired Student t-test, but is not limited to a specific test method and level of statistical significance.
  • the sample of the individual and the sample of the control group are biological samples, meaning all samples obtained from individuals whose p75, NCAM or CXCL13 protein of the present invention can be detected, and the biological samples include biopsy, blood, immune cells, It may be any one selected from the group consisting of nerve cells and skin tissues, preferably any one selected from the group consisting of serum, plasma, nerve cells, immune cells, cerebrospinal fluid, and exosomes, but is not particularly limited thereto, It can be prepared by processing in a method commonly used in the technical field of the present invention.
  • a method of measuring the concentration in a sample of mRNA which is a transcription material of a gene encoding p75, NCAM, or CXCL13 protein, or a concentration of the protein in a sample, may be selected, but is not limited thereto. It can be carried out by selecting a method commonly used in the technical field of the invention.
  • RT-PCR reverse transcriptase polymerase reaction
  • Competitive RT-PCR competitive reverse transcriptase polymerase reaction
  • Real-time RT-PCR real-time reverse transcriptase polymerase reaction
  • RNase protection assay RNase protection assay
  • the amount of protein may be determined using an antibody that specifically binds to the protein. Analysis methods for this include immunotaxonomy, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion, and rocket immunoelectricity. Electrophoresis, tissue immunostaining (immunohistochemistry), immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), FACS (fluorescence-activated cell sorting) and protein chip (protein chip), and the like, but are not limited thereto. .
  • the NCAM, p75, and CXCL13 may be those of the judgment object, for example, in the case of human, the mRNA sequence may be a sequence of SEQ ID NO: 1, 2, and 3, respectively, and the protein is, for example, SEQ ID NO: 4, 5, It may be the sequence of 6, but is not limited thereto.
  • the present invention provides a method for screening a candidate material for prevention or treatment for each subtype of peripheral neuropathy comprising the step of treating a test substance in a sample isolated from a peripheral neuropathy individual to compare the expression levels of p75 and NCAM proteins before and after the treatment. .
  • the method may further include the step of selecting a candidate material for preventing or treating peripheral neuropathy when the level of expression of p75 and NCAM protein is significantly reduced compared to before treatment of the test material.
  • the peripheral neuropathy may be hereditary peripheral neuropathy or non-hereditary peripheral neuropathy, and specific examples are Charcomaritus disease 1a (CMT1a), acute motor axonal neuropathy (AMAN), chronic inflammatory demyelination. It may be chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP).
  • CMT1a Charcomaritus disease 1a
  • AMAN acute motor axonal neuropathy
  • CIDP chronic inflammatory demyelinating polyradiculoneuropathy
  • AIDP acute inflammatory demyelinating polyradiculoneuropathy
  • the method further comprises the step of selecting a candidate material for preventing or treating hereditary peripheral neuropathy when there is no significant difference in the expression level of the p75 protein compared to before treatment of the test material, and the level of expression of the NCAM protein is significantly reduced.
  • the hereditary peripheral neuropathy may be Charcomaritus disease 1a (CMT1a).
  • the method may further include comparing the expression level of the CXCL13 protein before and after treatment with the test substance. In this case, when the expression levels of p75, NCAM and CXCL13 proteins are significantly reduced compared to before treatment with the test substance, it is inflammatory. It may further include the step of selecting as a candidate material for preventing or treating inflammatory demyelinating polyradiculoneuropathy.
  • the inflammatory demyelinating polymuscular neuropathy may include chronic or acute inflammatory demyelinating polymuscular neuropathy.
  • the test substance is a newly synthesized or known compound, and may include, without limitation, substances that are expected to exhibit an effect on the prevention or treatment of peripheral neuropathy subtypes.
  • nucleic acids, nucleotides, proteins, peptides, amino acids It may be at least one selected from the group consisting of sugars, lipids, and compounds, but is not particularly limited thereto.
  • the present invention is a peripheral neuropathy subtype comprising a nucleotide sequence of a gene encoding p75 and NCAM protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide or a substance that specifically binds to a protein encoded by the nucleotide sequence It provides a composition for star diagnosis.
  • the composition may be used for diagnosis of the onset of hereditary peripheral neuropathy or non-hereditary peripheral neuropathy, and specifically, the hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axon neuropathy.
  • CMT1a Charcomaritus disease 1a
  • AMAN Acute motor axonal neuropathy
  • CIDP Chronic inflammatory demyelinating polyradiculoneuropathy
  • AIDP acute inflammatory demyelinating polyradiculoneuropathy
  • the composition may further comprise a nucleotide sequence of a gene encoding CXCL13 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance that specifically binds to a protein encoded by the nucleotide sequence.
  • the composition of the case may be a composition for diagnosing inflammatory demyelinating polyradiculoneuropathy.
  • the substance specifically binding to the protein may specifically be an antibody, and the antibody refers to a specific immunoglobulin directed against an antigenic site, and the antibody is specifically directed to p75, NCAM or CXCL13 protein. It refers to an antibody that binds, and a gene encoding p75, NCAM or CXCL13 is cloned into an expression vector to obtain a p75, NCAM or CXCL13 protein, and an antibody can be prepared from the obtained protein according to a conventional method in the art.
  • the form of the antibody includes polyclonal antibody or monoclonal antibody, and all immunoglobulin antibodies are included.
  • the antibody is not only in complete form with two full-length light chains and two full-length heavy chains, but also has two light chains and two heavy chains, and does not have the structure of an antibody, but is directed against the antigenic site. It also includes functional fragments of antibodies molecules having a specific antigen-binding site (binding domain) and possessing an antigen-binding function.
  • binding domain binding domain
  • peripheral neuropathy can be diagnosed.
  • the selection and hybridization conditions of an appropriate antibody can be appropriately selected according to techniques known in the art.
  • the nucleotide sequence, a sequence complementary to the nucleotide sequence, and a substance that specifically binds to the fragment of the nucleotide may be specifically a probe or a primer.
  • the probe refers to a nucleotide fragment such as RNA or DNA corresponding to a few bases or hundreds of bases that can specifically bind to a nucleotide such as mRNA, and is labeled with a radioactive element, so that a specific mRNA exists. You can check the presence or absence and content (amount of expression).
  • the probe may be prepared in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe probe, etc., and encodes p75, NCAM or CXCL13 protein.
  • Peripheral neuropathy can be diagnosed by performing hybridization using a probe complementary to the mRNA of the gene, and measuring the expression level of the mRNA through the degree of hybridization. Selection of an appropriate probe and conditions for hybridization can be appropriately selected according to techniques known in the art.
  • the primer is a nucleotide sequence having a short free 3-terminal hydroxyl group, which can form a base pair with a complementary template, and refers to a short nucleotide sequence serving as a starting point for template strand copying.
  • the primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase/polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature, and p75, Peripheral neuropathy can be diagnosed through PCR amplification using a primer of an mRNA of a gene encoding NCAM or CXCL13 protein and measuring the expression level of a desired p75, NCAM or CXCL13 protein.
  • the PCR conditions and the length of the primer set may be appropriately selected according to techniques known in the art.
  • the nucleotide sequence of a gene encoding the p75, NCAM or CXCL13 protein, a sequence complementary to the nucleotide sequence, or a probe or primer that specifically binds to a fragment of the nucleotide is the nucleotide sequence of a gene encoding p75, NCAM or CXCL13 protein
  • a person skilled in the art can design the primer or probe according to a conventional method in the art based on the sequence.
  • the probe or primer can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known method, and the length is 10 to 100 nucleotides (hereinafter referred to as'nt'), 10 to 90 nt, 10 to 80 nt, 10 to 70 nt, 10 to 60 nt, 10 to 50 nt, 10 to 40 nt, 10 to 30 nt, 10 to 25 nt, 20 to 100 nt, 30 to 90 nt, 40 to 80 nt, 50 to 70 nt, 20 to 60 nt, 20 to 50 nt, 30 to 40 nt, 20 to 30 nt, or 20 to 25 nt.
  • a phosphoramidite solid support synthesis method or other well-known method, and the length is 10 to 100 nucleotides (hereinafter referred to as'nt'), 10 to 90 nt, 10 to 80 nt, 10 to 70 nt, 10 to 60 nt, 10 to 50
  • the present invention provides a diagnostic kit for each subtype of peripheral neuropathy comprising the composition.
  • the kit can diagnose peripheral neuropathy by measuring the expression level of p75, NCAM or CXCL13, the mRNA of the gene encoding the p75, NCAM or CXCL13 protein, or the expression level of the p75, NCAM or CXCL13 protein.
  • the peripheral neuropathy may be hereditary peripheral neuropathy or non-hereditary peripheral neuropathy, and specific examples are Charcomaritus disease 1a (CMT1a), acute motor axonal neuropathy (AMAN), chronic inflammatory demyelination. It may be chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP).
  • CMT1a Charcomaritus disease 1a
  • AMAN acute motor axonal neuropathy
  • CIDP chronic inflammatory demyelinating polyradiculoneuropathy
  • AIDP acute inflammatory demyelinating polyradiculoneuropathy
  • the kit may include a nucleotide sequence of a gene encoding a p75, NCAM or CXCL13 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance that specifically binds to a protein encoded by the nucleotide sequence.
  • the kit may include one or more other constituents/compositions, solutions, or devices suitable for an analysis method for measuring the expression level of p75, NCAM or CXCL13 protein used.
  • kits include test tubes or other suitable containers, reaction buffers, deoxyribonucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase, in addition to each pair of primers specific for the mRNA of the marker gene. Inhibitors, DEPC-water, sterile water, and the like. In addition, it may include a primer pair specific to the gene used as a quantitative control.
  • dNTPs deoxyribonucleotides
  • enzymes such as Taq-polymerase and reverse transcriptase
  • DNase reverse transcriptase
  • RNase reverse transcriptase
  • Inhibitors DEPC-water, sterile water, and the like.
  • it may include a primer pair specific to the gene used as a quantitative control.
  • the kit provides immunological detection of a nucleotide sequence of a gene encoding a p75, NCAM or CXCL13 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance that specifically binds to the protein encoded by the nucleotide sequence.
  • a substrate a suitable buffer solution, a secondary antibody labeled with a color developing enzyme or a fluorescent substance, and a color developing substrate may be included.
  • the substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass, and the color developing enzyme is peroxidase, alkaline phosphatase ( alkaline phosphatase) can be used, fluorescent materials can be used FITC, RITC, etc., and the color developing substrate is 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or o- Phenylenediamine (OPD), tetramethyl benzidine (TMB), and the like may be used.
  • ABTS 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)
  • OPD o- Phenylenediamine
  • TMB tetramethyl benzidine
  • the kit may be a microarray for peripheral neuropathy diagnosis capable of measuring the mRNA expression level of a p75, NCAM or CXCL13 protein or a gene encoding a protein thereof.
  • the microarray can be easily manufactured by a person skilled in the art according to a method known in the art, and according to one embodiment, the cDNA of the sequence corresponding to the mRNA of the gene encoding the p75, NCAM or CXCL13 protein or a fragment thereof is It may be a microarray attached to a substrate as a probe.
  • the kit may be a protein array or a protein chip for peripheral neuropathy diagnosis capable of measuring the expression level of p75, NCAM or CXCL13 protein.
  • the protein array or protein chip can be easily manufactured by a person skilled in the art according to a method known in the art, and according to one embodiment, p75, NCAM, or CXCL13 protein that is immobilized in the kit by extracting a sample from a patient Peripheral neuropathy can be diagnosed by subtype by confirming the reaction between a sample of a patient and a substance capable of measuring the expression level of p75, NCAM, or CXCL13 protein such as antibodies, receptors, nucleic acids, carbohydrates, etc. that can specifically bind to .
  • kits of the present invention matters related to the diagnostic composition, sample, or peripheral neuropathy included in the kit are as described above.
  • Antibodies against ⁇ -actin, p75 neurotrophin receptor (p75), CD68, CD4, CD63, Hsp70, and myelin basic protein (MBP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
  • NCAM and human CXCL13 antibodies were purchased from R&D Systems (Minneapolis, MN, USA).
  • Antibodies against CD206, Rab5b and CXCR5 were obtained from Abcam (Cambridge, UK), and the Alexa-Fluor 488 conjugated CD197 antibody was purchased from Biolegend (San Diego, CA, USA).
  • Antibodies against CXCL13 and myelin basic protein were obtained from Thermo Fisher Scientific (Waltham, MA, USA).
  • HRP Horseradish peroxidase binding anti-rabbit IgG and anti-mouse IgG were obtained from Cell Signaling technology (Danvers, MA, USA). Alexa Fluor 488 or Cy3 secondary antibody was purchased from Molecular probes (Carlsbad, CA, USA). All recombinant cytokines used were obtained from Peprotech (Rocky Hill, NJ, USA) and R&D Systems, and unless otherwise specified, all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
  • Non-obesity diabetes (NOD) and NOD-B7-2 knockout (B7-2KO) mice were purchased from Jackson Lab (Bar Harbor, Stock No. 004762, USA). The genotype was determined, and neuropathy was evaluated weekly from 20 weeks after birth. Tail-drop and hind-limb were investigated. The clinical progression of motor deficit was divided into 5 grades: Grade(G)0, no symptoms; G1, floppy tail; G2, mild paraparesis or unilateral hind limb paralysis; G3, severe catastrophic paralysis; G4, tetraparesis; G5, dying condition or death.
  • PMP22 transgenic mice (C22) 19 were obtained from Samsung Medical Center (Seoul, Korea).
  • the mouse model contains seven human peripheral myelinated protein 22 (PMP22) genes that cause demyelinating neuropathy. All surgical operations were performed according to the animal testing guidelines established by Dong-A University Animal Testing Society (No. DIACUC-16-21) and the protocol approved by Dong-A University Animal Testing Committee.
  • PMP22 peripheral myelinated protein 22
  • distal stumps of 1 mm length from the lesion site were discarded, and distal stumps of 5 mm length were collected at the indicated time.
  • Serum samples included 36 CIDPs (10 females, 26 males), 14 AIDPs (3 females, 11 males), 20 AMANs (7 females, 13 males) and 39 CMT1a (17 females, 21 males) patients and 20 healthy controls (14 females, 6 males).
  • the blood was centrifuged at 3000 rpm for 10 minutes to separate the serum (plain tube, no anticoagulant), and the collected serum was stored at -80°C until use.
  • the diagnosis of CIDP and GBS (AIDP, AMAN) was performed according to clinical and laboratory criteria, respectively.
  • AMAN was classified according to the positive anti-ganglioside GM1 antibody using ELISA.
  • Sciatic nerve sections were taken at 37° C. for 80 minutes, collagenase NB4 (0.26 U/ml, Serva, Heidelberg, Germany) and dispase ⁇ (neutral protease, grade ⁇ , 0.94 U/ml, Roche, CA, USA). ) was digested with an enzyme solution containing.
  • the mixture was centrifuged at 1000 rpm for 10 minutes, and the supernatant was removed, and then the cell pellet was neuregulin-1 (neuregulin-1, 30 ng/ml, R&D Systems), N2 supplement (Invitrogen, Carlsbad, CA), 5 ⁇ M It was cultured in a medium containing forskolin, 1% fetal bovine serum (FBS, Hyclone, Melbourne, Australia), and penicillin-streptomycin (Gibco, NY, USA). After 48 hours, cells were treated with 0.2% dispase ⁇ diluted in DMEM for 20 minutes, and then kept by shaking horizontally for 1-3 minutes to enrich the SC in the culture flask.
  • neuregulin-1 neuregulin-1, 30 ng/ml, R&D Systems
  • N2 supplement Invitrogen, Carlsbad, CA
  • 5 ⁇ M It was cultured in a medium containing forskolin, 1% fetal bovine serum (FBS, Hyclone, Melbourne
  • the suspended cells were collected by centrifugation at 1000 rpm for 5 minutes, and after removing the supernatant, the pellet was resuspended and plated on a flask at a density of 2 to 2.5 x 10 4 cells/cm 2.
  • cells were used in 2-4 generations.
  • SCs were incubated with DMEM containing 5 ⁇ M forskolin, 30 ng/mL neuregulin-1, and 1% exosome-free FBS (obtained by serum ultracentrifugation at 100,000 g for 12 hours). After incubation for 3 days, the culture solution was collected and continuously centrifuged at 4° C. for 30 minutes at 300 g and 60 minutes at 10,000 g, and the supernatant was filtered through a membrane filter (0.2 ⁇ m, Sartorius Biotech, Goettingen, Germany), and 4° C. Ultracentrifugation was performed at 100,000 g for 90 minutes at (70 Ti rotor, Beckman).
  • the pellet was washed with phosphate buffered saline (PBS, pH 7.4) and centrifuged again for 60 minutes at 100,000 g at 4°C.
  • Purified exosomes were modified with 1% phenylmethylsulfonylfluoride and 1% protease inhibitor cocktail (Sigma-Aldrich) in RIPA buffer (1% Triton X-100, 50 mM Tris-HCl, pH 6.8, 2 mM EDTA) Dissolved in, and quantified by Bradford assay or microBCA assay (Thermo Fisher Scientific).
  • Proteins in exosomes were separated by electrophoresis in precast Bolt TM 4-12% Bis-Tris Plus SDS-PAGE Gel (Thermo Fisher Scientific) and stained with Coomassie Brilliant Blue R-350 (GE Healthcare, Uppsala, Sweden).
  • the gel lane is excised into 14 sections, and then cut into 1-2 mm cubes with a clean scalpel.
  • the gel pieces were washed twice with 30% methanol and removed in 50% acetonitrile in 100 mM ammonium hydrogen carbonate.
  • the sample was then reduced with 10 mM DTT for 1 hour at 56° C. and alkylated with 20 mM iodoacetamide for 1 hour at room temperature in the dark.
  • Trypsin-degrading peptides derived from Schwann cell exosomes are easy nLC 1000 coupled online with reversed-phase nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) (LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific)). system (Thermo Fisher Scientific)).
  • the eluent was sprayed and ionized at 1.9 kV in a nano-electron spray source of a mass spectrometer operated in a data dependent acquisition mode.
  • MS survey scans were performed in an orbitlab with a resolution of 60,000 FWHM (200 m/z) over a mass range of 400-2,000 m/z, followed by a collision energy of 35%, an activation time of 10 ms, and a linear trap quadrupole. trap quadrupole, LTQ) collision-induced dissociation MS/MS fragmentation was performed on the fifteen most intense ions with an isolation window of 2 Da. For dynamic exclusion, the number of repetitions was set to 2 and the exclusion period was set to 60 seconds.
  • Schwann cells cultured for western immunoblotting were homogenized in modified RIPA buffer.
  • the lysate was fractionated on an SDS-PAGE gel and transferred to a nitrocellulose membrane (Millipore).
  • Primary antibody diluted in TBST containing 3% fat-free milk for 1 hour at room temperature with 5% skim milk in Tris buffered saline containing 0.05% Tween-20 (TBST) and the membrane at 4°C overnight was incubated with. After washing three times in TBST, it was reacted with HRP-conjugated secondary antibody for 1 hour at room temperature, and then washed again with TBST.
  • cytokine antibody array designed to monitor the expression level. Briefly, lysates (300 ⁇ g) were incubated overnight with an antibody-coated membrane on an incubated platform, washed three times with PBS to remove unbound material, and then aligned capture antibody and biotinylated ( biotinylated) detection antibodies were used to continuously detect bound cytokines via a sandwich ELISA format. After washing three times with PBS, chemiluminescence detection reagent (Amersham) was added, and then proceeded according to the manufacturer's instructions.
  • Results were expressed as mean ⁇ standard error, and ELISA data were analyzed by one-way analysis of variance by Kruskal-Wallis test and Sidak's multiple comparisons test using GraphPad Prism version 6.01 (GraphPad Software Inc., La Jolla, CA). . An unpaired student t-test was performed for the Western blot and statistical analysis of the number of immune response cells, and a value of p ⁇ 0.05 was considered significant.
  • the protein in the exosomes derived from mouse Schwann cells included p75, NCAM and well-known immature Schwann cell proteins, and significant enrichment of p75 and NCAM proteins in mouse Schwann cell-derived exosomes was confirmed through western blot. (Fig. 1a C).
  • p75 and NCAM in the serum of various peripheral neuropathy patients were examined using ELISA (Fig. 1B D, E). At least 3 times higher p75 in serum from CIDP (256 ⁇ 31.26 pg/mL, p ⁇ 0.001) and AIDP (207.3 ⁇ 37.85 pg/mL, p ⁇ 0.001) mL) patients compared to healthy controls (73.69 ⁇ 40.58 pg/mL) Indicated the concentration.
  • the serum NCAM concentrations of patients with CIDP (4,960 ⁇ 476 pg/mL, p ⁇ 0.001) and AIDP (4,729 ⁇ 661 pg/mL, p ⁇ 0.05) were also higher than that of the healthy control group (2,298 ⁇ 303 pg/mL).
  • the serum concentration of NCAM and p75 was higher than that of the control group, but it was not significant.
  • the highest level of NCAM concentration (6,663 ⁇ 277 pg/mL) was observed, while the p75 concentration (12.74 ⁇ 5.315 pg/mL) was similar to that of the control group.
  • the center dotted line is the result of CIDP
  • the NCAM of G the result of CMT1a of FIG. 1BE
  • the center dotted line is the result value of CMT1A.
  • DSC demyelinating SC
  • EAN experimental allergic neuritis
  • WD Wallerian degeneration
  • axonal cleavage did not induce the expression of NCAM in DSC, and did not significantly increase the expression of NCAM in non-myelinating SC (FIGS.
  • the pro-inflammatory state-related NCAM expression in dedifferentiated SC was induced to compare the cytokine expression profile in the neuropathic sciatic nerve and the damaged C57BL/6 nerve in B7-2KO mice (in this case, natural immunity was observed during WD. Activated).
  • Expression levels of ICAM-1, interleukin-1 receptor antagonist, metalloproteinase 1 tissue inhibitor, CCL2 and IL-16 were increased in both the sciatic nerve of B7-2KO mice and injured C57BL/6 mice, and CCL5, macrophages It was found that inflammatory protein-1 and CXCL10 were specifically upregulated in B7-2KO neurons compared to NOD and damaged C57BL/6 mouse neurons (Figs. 5A A, 5A B).
  • CCL5 and CXCL10 are cytokines associated with INF- ⁇ -induced type-1 macrophage (M1), and both are known to be involved in inflammatory neuropathy.
  • CXCL13 a B cell recruitment factor, is constitutively expressed in follicular cells and macrophages in secondary lymph node tissues, and is selectively upregulated in B7-2KO neurons, but not in WD (Figs. 5A A, 5A B).
  • the demyelinating B7-2KO neuron exhibits a pro-inflammatory environment associated with M1-macrophages, which is associated with selective CXCL13 and NCAM expression in inflammatory demyelination.
  • CXCL13 in B7-2KO neurons may indicate local B cell-related immune activation through CXCR5, a receptor for CXCL13.
  • Invasion of CD19+ B cells in the B7-2KO and NOD nerves was investigated. It was found that B cells penetrated the B7-2KO nerve a lot, but this was not the case in the NOD nerve (Fig. 6A).
  • the cellular localization of CXCR5 in the B7-2KO nerve was examined using IF staining. The expression level of CXCR5 was low in the axon and SC of the NOX nerve, but the expression of CXCR5 was rapidly increased in the B7-2KO nerve.
  • Double immunostaining showed that many of the CXCR5 positive mononuclear cells in B7-2KO neurons were CD4+ T cells and CD68+ macrophages (Figs. 6A A, 6B B).
  • some p75 positive dedifferentiated SCs also express CXCR5 in B7-2KO neurons (Fig. 6BB).
  • Expression of CXCR5 in the SC was confirmed by Western blot analysis in the cultured primary SC (FIG. 6CC), which is the pathology of CXCL13 and CXCR5/CD4+ T cells, B cells and SC in autoimmune inflammatory demyelination in mice. This suggests that it could be involved as an enemy.
  • Serum CXCL13 levels of patients with peripheral neuropathy were examined. Serum of patients with CIDP (71.41 ⁇ 5.83 pg/mL, p ⁇ 0.01) and AIDP (71.34 ⁇ 13.73 pg/mL, p ⁇ 0.05) patients with control (37.35 ⁇ 6.75 pg/mL) mL) of serum and found a higher level of CXCL13. In contrast, CXCL13 levels in serum of AMAN (17.99 ⁇ 7.02 pg/mL) and CMT1a (47.39 ⁇ 3.98 pg/mL) were not significantly different from those of control (p>0.05, Fig. 7).
  • CXCL13+ cell infiltration was found in the gastrocnemius in CIDP patients, but not in CMT1a patients. These results indicate that CXCL13 may be a specific marker of inflammatory peripheral demyelination.
  • ELISA data showed a high level of NCAM in the serum of CMT1a patients (Fig. 1).
  • Since immature SC generally expresses NCAM at the beginning of birth, the expression of Pmp22 was wrong, resulting in NCAM expression of many abnormally differentiated immature SCs. This may be associated with high NCAM levels in the serum of CMT1a patients.
  • the expression of NCAM in the sciatic nerve of C22 mice which is a PMP22 overexpressing CMT1a mouse model, was investigated. Mice showed abnormal gait at 2 weeks of age and were maintained until the mice were sacrificed. The sciatic nerve was not well myelinated at 5 and 8 weeks after birth in C22 mice (Fig. 8A A).
  • NCAM immune responsive cells were found more frequently in the sciatic nerve of C22 mice compared to the control nerve (Figs. 8AB, 8BC). High levels of NCAM expression in C22 neurons were also confirmed by Western blot analysis (Fig. 8BC). Most of the NCAM staining in the C22 nerve was patch-shaped, but it was found to be different from the abaxonal perimyelin staining of demyelinating SC in B7-2KO mice (Fig. 8A B), and the large number of NCAM-positive cells in the C22 nerve was extra immature SC This suggests that may be the cause (Figs. 8AB, 8BC).
  • the number of DAPI-positive cells was about 2 times higher in the C22 sciatic nerve (786 ⁇ 73, p ⁇ 0.001) compared to the WT control (322 ⁇ 26), but macrophage infiltration was mild (45.7 ⁇ 5.6 [C22]). 26.3 ⁇ 11.7 [WT], p ⁇ 0.001, Fig. 8BD), mostly CD206+/CXCL13-.
  • NCAM expression was found not only in a number of dot-shaped non-myelinated SCs, but also in all the SC layers of onion-bulb (FIG. 8C E ).

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Abstract

La présente invention concerne un procédé pour fournir des informations pour prédire ou diagnostiquer un risque de développer chaque sous-type de neuropathie périphérique à l'aide d'une combinaison de biomarqueurs ; une composition pour diagnostiquer chaque sous-type ; et un procédé de criblage de médicament pour traiter une neuropathie périphérique. Le procédé de la présente invention mesure un niveau d'expression d'un biomarqueur pour fournir efficacement des informations pour prédire ou diagnostiquer le risque de développer chaque sous-type de neuropathie périphérique, et la composition et le kit de diagnostic de la présente invention peuvent diagnostiquer des sous-types spécifiques de neuropathie périphérique avec une précision élevée.
PCT/KR2019/016923 2019-04-12 2019-12-03 Biomarqueur pour diagnostiquer chaque sous-type de neuropathie périphérique Ceased WO2020209469A1 (fr)

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