KR102513533B1 - Composition for diagnosis of charcot-marie-tooth disease and method for providing information for diagnosis tehreof - Google Patents

Abstract

The present invention relates to a composition for diagnosis of Charcomaritus disease and a method for providing information for diagnosis thereof. By detecting a p62 protein in serum or plasma, measuring the concentration, and comparing the measured concentration with that of a control group, the method can be usefully used for diagnosing and determining the severity of the Charcomaritus disease.

Description

Composition for diagnosis of Charcotosis disease and method for providing information for diagnosis thereof

The present invention relates to a composition for diagnosing Charcomaritus disease and a method for providing information for diagnosis.

Charcot-Marie-Tooth disease (CMT), also known as hereditary motor and sensory neuropathy, is a progressive sensory impairment and muscle weakness. Charcomaritus disease is characterized by muscle weakness and atrophy that gradually progresses from the distal end to the proximal part of the lower limbs, with areflexia, distal sensory loss, pes cavus, and hearing loss. Deafness may also appear. Onset usually occurs around the age of 10, but rarely occurs after the age of 30. About 120 causative genes of Charcomaritus disease are known to date, and even if symptoms occur due to mutations in the same gene, the clinical phenotype and age of onset are very diverse, making diagnosis difficult if there is no family history. Nerve biopsy can be helpful in diagnosis, but it is not easy to use for diagnosis as an invasive test.

Recently, as next generation sequencing (NGS) is actively used for disease diagnosis, research to isolate the causative gene and identify molecular biological mechanisms is being actively conducted. amounts to about 30% of In addition, as clinical research on gene therapy for Charcomaritus disease is currently underway, the importance of accurate diagnosis of Charcomaritus disease is being emphasized.

In addition, there are various techniques for evaluating the severity of patients' symptoms after diagnosis of Charcomaritus disease, but most of them are expensive or very cumbersome methods such as MRI or electrical conduction test, and a simple but accurate evaluation technique using serum or plasma is required. In particular, we are trying to develop a method for evaluating the improvement in severity after customized treatment for Charcomaritus disease, but there is no universal method.

Berger, P., Young, P. & Suter, U. Molecular cell biology of Charcot-Marie-Tooth disease. Neurogenetics 4, 1-15 (2002)

The clinical phenotype and onset age of Charcomaritus disease are very diverse, and about 30% or more of patients do not have a family history or do not receive an accurate diagnosis even through next-generation sequencing. Nerve biopsy is also an invasive test, and there are limitations in using it as a diagnostic test every time.

Accordingly, research on new biomarkers capable of accurately diagnosing Charcomaritus disease and determining the severity of the patient's symptoms is being conducted. A biomarker is an indicator that can detect changes in the body using proteins, DNA, RNA, metabolites, etc. Using biomarkers, it is possible to objectively measure the normal or pathological state of a subject, the degree of response to drugs, etc. can Biomarkers are called endogenous phenotypes of a specific disease and can be used to find the cause of a specific disease or measure the risk of developing it.

In particular, research is being conducted to utilize a gene (TMPRSS5) specifically expressed in neurofilament (NFL) or Schwann cells, which is highly detected in patients with Charcomaritus disease compared to non-patients, as a biomarker.

However, cases in which protein biomarkers using non-invasively obtainable serum or plasma as in the present invention are used for diagnosis of Charcomaritus disease are extremely rare. In particular, previously reported serum diagnostic aid proteins are present in the serum of non-patient controls, making it difficult to have diagnostic value. Therefore, the development of protein biomarkers for simple and highly accurate diagnosis of Charcomaritus disease through serum or plasma is required.

1. A composition for diagnosing Charcomaritus disease, comprising an agent for detecting the expression of p62 protein in serum or plasma.

2. The composition for diagnosing a Charcomaritus disease according to 1 above, wherein the Charcomaritus disease is selected from the group consisting of a demyelinating type, an axonal type, and an intermediate type.

3. A kit for diagnosing Charcomaritus disease comprising the composition of any one of 1 or 2 above.

4. A method for providing information for diagnosing Charcomaritus disease, which provides information that the individual has a higher probability of developing Charcomaritus disease than the control group when the concentration of p62 protein in serum or plasma isolated from the subject is higher than that of the control group.

5. The method of providing information for diagnosing Charcomaritus disease according to 4 above, wherein the control group is a non-Charcomarithmic disease patient.

6. A method for providing information for diagnosing Charcomaritus disease, providing information that the individual has developed Charcomaritus disease when the concentration of p62 protein in serum or plasma isolated from the individual is higher than 229.4 pg/ml.

7. If the concentration of p62 protein in serum or plasma isolated from a patient with Charcomitus disease is higher than that of the control group, the diagnosis of Charcomitus disease, which provides information that the patient is more likely to have severe Charcomitus disease than the control group How to provide information for

The diagnostic composition and kit of the present invention can diagnose Charcomaritus disease in a non-invasive way.

The method of the present invention can provide information for diagnosing or determining the severity of Charcomaritus disease in a non-invasive and simple manner.

The composition/method of the present invention can diagnose Charcomaritus disease with high accuracy or provide information necessary for diagnosis.

1 is an experimental result of the p62 protein expression level in patients with Charcomaritus disease compared to non-patients with Charcomaritus disease.
Figure 2 shows the results of testing the p62 protein expression level according to the severity, onset period, and age of Charcomaritus disease.
3 is an experiment result of the p62 protein expression level for each severity in different subtypes according to gene types of Charcomaritus disease.
4 is an experimental result of p62 protein expression in muscle cells of C22 mice compared to WT mice.
5 is an experimental result of p62 protein expression in sciatic nerve and Schwann cells of C22 mice compared to WT mice.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for diagnosing Charcomaritus disease.

The composition for diagnosing Charcomaritus disease of the present invention includes an agent for detecting the expression of p62 protein in serum or plasma.

The present inventors conceived the present invention based on the finding that the concentration of p62 protein in serum or plasma is positively correlated with the occurrence of Charcomaritus disease, and that the higher the concentration, the higher the severity of the disease.

The composition of the present invention includes an agent for detecting the expression of p62 protein in serum or plasma, so that it can be used to measure the presence or absence of p62 protein in serum or plasma and its concentration, thereby diagnosing the occurrence of Charcomaritus disease or , can be used to diagnose its severity.

For example, when the concentration of p62 protein in the serum or plasma of a patient suspected of Charcomaritus disease is compared with the concentration in a non-patient control group, when the concentration of p62 protein in the serum or plasma of a patient suspected of Charcomaritus disease is higher, the non-patient control group It can be diagnosed that the probability of developing Charcomaritus disease is higher than that. Alternatively, by comparing the concentration of p62 protein in the serum or plasma of two suspected patients, it can be diagnosed that a patient with a higher concentration of the protein is more likely to develop Charcomaritus disease or has more advanced symptoms.

The type of agent for detecting protein expression in serum or plasma is not limited as long as it can be detected by interacting with p62 by binding to or reacting with the p62 protein in serum or plasma. For example, it may be an antibody, aptamer, protein, peptide, nucleic acid, polymer, compound or reagent.

Charcomaritus disease may be selected from the group consisting of demyelinating, axonal and intermediate types. For example, it may be CMT1A among demyelinated types, CMT2A among axonal types, or CMTX1 among intermediate types, but is not limited thereto.

In addition, the present invention relates to a kit for diagnosing Charcomaritus disease comprising the composition.

The kit may include a material that specifically binds or reacts to the p62 protein, and may include one or more additional compositions, configurations, devices, etc. suitable for an assay method for measuring the protein expression level.

As analytical methods, for example, BCA (Bicinchoninic acid) quantification method, Biuret reaction quantification method, Lowry test (Folin-Ciocalteau), Bradford quantification method, ultraviolet spectrophotometry, eg Warburg-Christian method or Waddell method, Micro-Kjeldahl quantification method Or a combination thereof may be used, but is not limited thereto, and protein assays commonly used in the art may be used.

The kit includes an antibody that specifically binds to the p62 protein, a secondary antibody conjugate conjugated with a label that develops color by reaction with the substrate, a color-developing substrate solution to react with the label, a washing solution, and an enzyme reaction stopping solution. and the like, and may be manufactured in a number of separate packaging or compartments containing reagent components to be used, but is not limited thereto.

In addition, the kit may include, but is not limited to, a substrate, an appropriate buffer solution, a secondary antibody labeled with a detection label, and a chromogenic substrate for immunological detection of the antibody.

As a specific example, the kit may be a kit characterized in that it includes essential elements required to perform ELISA in order to implement various ELISA methods such as an ELISA kit and a sandwich ELISA kit. These ELISA kits contain antibodies specific for the protein. The antibody has high specificity and affinity for the P62 protein and little cross-reactivity to other proteins, and may be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. ELISA kits may also include antibodies specific for a control protein. Other ELISA kits may include reagents capable of detecting bound antibodies, for example, labeled secondary antibodies, chromophores, enzymes and their substrates, or other substances capable of binding to antibodies, etc. may not be limited thereto.

In addition, the kit may be a kit for implementing Western blot, immunoprecipitation assay, complement fixation assay, flow cytometry, or protein chip, and may further include additional components suitable for each assay method. Through these analysis methods, Charcomaritus disease can be diagnosed by comparing the amount of antigen-antibody complex formation.

In addition, the present invention relates to a method for providing information for diagnosing Charcomaritus disease.

According to the method of the present invention, when the concentration of p62 protein in serum or plasma isolated from an individual is higher than that of a control group, information indicating that the individual has a higher probability of developing Charcomaritus disease than the control group is provided.

The subject is an animal suspected of having Charcomatith disease, an animal to which information for diagnosing Charcomaritus disease is desired, and the animal may be a mammal including a human.

The control group may be, for example, a non-patient of Charcomaritus disease, and the serum or plasma p62 protein concentration of the subject is compared with their serum or plasma p62 protein concentration, or the average value, median value, etc. of a group including them is compared. may be

Since Charcomaritus disease patients have a higher serum or plasma p62 protein concentration than other cases, if the serum or plasma p62 protein concentration of the individual is higher than that of the control group, the probability of the individual developing Charcomaritus disease compared to the control group is higher. Higher information can be provided.

The method of the present invention may further include measuring the concentration of p62 protein in serum or plasma isolated from the subject and comparing it with the concentration in a control group.

The concentration measurement may be performed using, for example, the aforementioned composition, and methods known in the art used for measuring protein concentration may be used without limitation.

For the concentration comparison, methods known in the art may be applied without limitation.

In addition, the present invention relates to a method for providing information for diagnosing Charcomaritus disease, which provides information that the individual has developed Charcomaritus disease when the concentration of p62 protein in serum or plasma isolated from the individual is higher than 229.4 pg/ml. will be.

The serum or plasma p62 protein concentration value of 229.4 pg/ml is a cut-off value for distinguishing between a Charcomaritus disease patient and a non-patient group. It can provide information that Maritus disease has developed.

The method of the present invention may further include the step of measuring the concentration of p62 protein in serum or plasma isolated from the individual, which is described above.

In addition, the present invention is a Charcomaritus disease diagnosis that provides information that the patient is more likely to have a severe Charcomaritus disease than the control group when the p62 protein concentration in serum or plasma isolated from a Charcomitus disease patient is higher than that of the control group. It is about how to provide information for

The Charcomatith disease patient is an animal with Charcomatith disease, an animal to which information for diagnosis of Charcomatith disease is sought, and the animal may be a mammal including a human.

The control group may be, for example, a Charcomaritus disease patient, and the serum or plasma p62 protein concentration of the individual is compared with the serum or plasma p62 protein concentration of the control patient, or the average value, median value, etc. of the control patients is compared. may be

The severity of Charcomaritus disease can be divided according to the degree of manifestation of symptoms caused by the disease, and the higher the degree of symptom expression, the higher the severity of Charcomaritus disease. Such conditions may include, for example, distal muscle weakness and atrophy, neurogenic muscular atrophy, inhibition of autophagy in muscle, denervation-induced muscle loss, progressive muscle wasting, and the like.

For example, the severity of Charcomaritus disease can be measured using CMT neuropathy score version 2 (CMTNSv2), CMTNSv2 ≤ 10 is mild, CMTNSv2 is 11-20, moderate, and CMTNSv2 ≥ 21 is severe. can be divided into three groups of The Charcomaritus disease scoring system is not limited thereto, and systems such as CMTPedS, CMT-FOM, or CMTInfS may be used.

Charcomaritus disease patients have a higher serum or plasma p62 protein concentration than other cases, and the higher the degree of expression of the symptoms, for example, the higher the CMTNSv2 value, the higher the serum or plasma p62 protein concentration. If the p62 protein concentration in the patient's serum or plasma is higher than that of the control group, information that the patient's Charcomaritus disease is more likely to be severe than the control group can be provided.

The method of the present invention may further include measuring the concentration of p62 protein in serum or plasma isolated from the patient and comparing it with the concentration in a control group.

The concentration measurement may be performed using, for example, the aforementioned composition, and methods known in the art used for measuring protein concentration may be used without limitation.

For the concentration comparison, methods known in the art may be applied without limitation.

Hereinafter, examples will be described in detail to explain the present invention in detail.

Example. Identification of CMT patients from normal controls and classification of CMT patients by severity based on difference in plasma p62 level measured by ELISA

The difference in plasma p62 levels between CMT patients and healthy controls (HC) was compared by ELISA using the p62-containing biomarker composition of the present invention. In this Example, in order to determine whether the plasma p62 concentration can be altered in CMT patients in consideration of p62 secretion in pathological conditions, ELISA was performed to measure plasma p62 levels in CMT patients and age-matched healthy controls. In addition, we sought to identify potential sources of plasma p62 using an animal model of CMT1A as well as correlating plasma p62 concentrations with various clinical parameters of CMT subtypes. A more detailed experimental procedure is described below.

1. Test subject and method

(1) Participants and study design

From 2020 to 2021, 138 CMT patients were consecutively enrolled after approval from the Department of Neurology and Genetic Neuropathy Clinic of Samsung Seoul Hospital. Written informed consent was obtained from all participants according to a protocol approved by the Bioethics Committee (IRB) of Sungkyunkwan University Samsung Medical Center (SMC, 2020-01-146). PMP22 duplication leading to CMT1A was determined by hexaplex microsatellite PCR for the chromosome 17p12 region. Other mutations were screened for using whole exome sequencing and confirmed using Sanger sequencing. All CMT patients had confirmatory causal gene mutations (Table 1). Disease severity was measured using CMTNSv2. CMT patients were divided into three groups according to the severity of the disease: mild (CMTNSv2 ≤ 10), moderate (CMTNSv2, 11-20), and severe (CMTNSv2 ≥ 21).

Demographic data of CMT patients and HC, median p62 plasma concentration, CMTNSv2 and INCAT scores Patient group n Age(y) (SEM) Sex, F/M Duration (SEM) p62, pg/mL (IQR) CMTNSv2 (IQR) INCAT (IQR) CMT 138 44.90 (1.174) 58/80 22.67 (1.372) 2354 (1603-3622) 14.0 (9-18) NA Control 59 44.88 (1.069) 30/29 NA 1978 (1139-2522) NA NA Demyelinating CMT CMT1A 70 47.26 (1.544) 34/36 21.66 (2.101) 2520 (1820-3958) 13 (8-18) NA CMT1B 6 43.5 (6.776) 3/3 31.67 (8.739) 1957 (1486-2166) 16.5 (14.25-18.75) NA CMT1C One 56 1/0 16 2698 16 NA CMT1E 8 39.38 (6.118) 4/4 25.13 (5.761) 1781 (1637-3555) 12.5 (8.25-19.75) NA CMT4B3 2 63 (2) 2/0 55 (1) 4218 (3756-4681) 24 NA CMT4F One 45 0/1 45 4269 32 NA CMT4H One 26 0/1 20 4666 18 NA Axonal CMT CMT2A 11 46 (3.737) 6/5 29.27 (3.231) 1739 (1212-2544) 15 (10-20) NA CMT2EE One 31 0/1 18 3597 18 NA CMT2F 2 37.5 (5.5) 0/2 16 (4) 899 (788-1011) 10.5 (10-11) NA CMT2O One 45 0/1 20 3459 17 NA CMT2S One 33 0/1 One 2282 10 NA CMT2U One 63 1/0 5 5203 19 NA Other CMT2 ^a 3 44.0 (5.686) 0/3 17 (6.11) 3735 (403-3816) 11 (10-18) NA Medium CMT CMTX1 25 39.76 (2.936) 5/20 18.85 (2.153) 2123 (1509-3395) 13 (9.5-16.5) NA CMTDIC One 59 1/0 42 6004 19 NA CMTDIF One 20 1/0 14 381 13 NA CMTDIG One 52 0/1 35 1230 14 NA Int-CMT ^b One 49 0/1 20 3695 18 NA

Abbreviations: CMTNSv2 = Charcot-Marie-Tooth neuropathy score version 2; INCAT = Inflammatory neuropathy cause and treatment disability score; CMT1 = demyelinating neuropathy CMT; CMT1A = PMP22 duplication; CMT1B = MPZ mutation; CMT1C = LITAF mutation; CMT1E = PMP22 point mutation; CMT4B3 = SBF1 mutation; CMT4F = PRX mutation; CMT4H = FGD4 mutation; CMT2A = MFN2 mutation; CMT2EE = MPV17 mutation; CMT2F = HSPB1 mutation; CMT2O = DYNC1H1 mutation; CMT2S = IGHMBP2 mutation; CMT2U = MARS mutation; CMTX1 = GJB1 mutation; CMTDIC = dominant intermediate CMT type C, YARS mutation; CMTDIF = GNB4 mutation; CMTDIG = NEFL mutation; SEM = standard error of the mean; IQR = interquartile range; NA = not applicable

^a Other CMT2: axonal neuropathy CMT2 with KIF5A or RAB40B mutations

^b Int-CMT: intermediate CMT neuropathy with DRP2 mutation

For plasma collection from HC, we visited Dong-A University Hospital for health check-up and enrolled subjects whose sex and age were matched to the CMT group. Exclusion criteria included anemia that could contribute to peripheral neuropathy, liver or kidney damage, and elevated fasting plasma glucose levels. Those with neurological symptoms or history-based neurological disease were excluded.

(2) Antibodies and reagents

Antibodies against p62, glyceraldehyde 3-phosphate dehydrogenase and myelin basic protein (MBP) were purchased from Abcam (Cambridge, UK). Antibodies against medium-sized neurofibrillary tangle (NF-M) and β-actin were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and Santa Cruz biotechnology (CA, USA), respectively. Horseradish peroxidase (HRP)-linked anti-rabbit IgG was purchased from Cell Signaling Lab (Beverly, MA, USA). Alexa Fluor 488 or Cy3-conjugated secondary antibodies were purchased from Molecular probes (Carlsbad, CA, USA). Unless otherwise specified, all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

(3) Blood sample collection and storage

Blood samples were taken and processed within 1 hour. Blood was collected in an SST tube, centrifuged at 3,000 rpm for 10 minutes at 4°C, and plasma was aliquoted and stored at -80°C.

(4) standard protocol approval, registration and patient consent

The research protocol was Dong-A University Bioethics Committee (No. HR-004-02), Dong-A University Hospital (No. 13-042), Inje University Busan Paik Hospital (No. 2015-01-271), Samsung Seoul Hospital (No. 2020 -01-146) was approved.

(5) Measurement of p62

Plasma p62 was measured using a commercially available ELISA kit (LSbio, LS-F49427, Seattle, USA). All samples were analyzed in triplicate without dilution according to the manufacturer's instructions. Samples with a mean p62 concentration of 2354 pg/ml had an average repeatability coefficient of variation of 7.31% and an assay-to-assay coefficient of variation of 8.78%. The limit of detection determined by the average blank signal +3 SD for the p62 assay was 51.19 pg/ml. The lower limit of quantification (LLOQ), measured as mean blank signal +10 SD, was 173.5 pg/ml. All samples in which p62 was detected under LLOQ were considered to have a zero concentration.

(6) Animal testing

All animal experiments were performed according to protocols approved by the Animal Research Committee of Dong-A University (No. DIACUC 21-8). C22 mouse [B6; CBACa-Tg(PMP22)C22Clh/H] was purchased from Samsung Medical Center. The mouse model contains seven copies of the human Pmp22 gene that causes demyelinating neuropathy.

For sciatic nerve injury, the left sciatic nerve of adult C57BL/6 mice was treated with 10% ketamine hydrochloride (Sanofi-Ceva, Duesseldorf, Germany; 0.1 ml/100 g body weight) and Rompun (Bayer, Leverkusen, Germany; 0.05 ml/100 g body weight). g body weight) mixture, and axons were severed at 5 mm proximal to the bifurcation of the tibia with fine iris scissors (FST Inc, Foster City, CA) (FST Inc, Foster City, CA). For neurodegenerative analysis, a 1 mm long distal amputee was discarded from the lesion site and the next 5 mm long distal amputee was collected at the indicated time points.

(7) Immunofluorescence (IF) staining and myocyte area measurement

Control C57BL/6 mice and C22 mice were perfusion-fixed with 4% paraformaldehyde dissolved in phosphate buffered saline (PBS), and the sciatic nerve and gastrocnemius muscle were harvested and cryoprotected in 20% sucrose solution. For p62 expression analysis, sections with a thickness of 10 μm were prepared using a cryocut and stored in a freezer until use. Slides were blocked in PBS containing 0.2% Triton X-100 and 5% bovine serum albumin for 1 hour at room temperature. Next, the slides were incubated with the primary antibody overnight at 4°C and then washed three times with PBS. The slides were then incubated with Cy3- or Alexa 488-conjugated secondary antibodies at room temperature for 3 hours and stained with DAPI for 30 minutes. For light microscopy analysis, stained sections were examined with an ApoTome fluorescence microscope with a Zeiss AxioImager 2 or Image Express confocal microscope (Carl Zeiss, Goettingen, Germany).

To measure the cytoplasmic area of myocytes with or without p62-positive spots in the gastrocnemius muscle, p62-positive myocytes were defined as myocytes with 5 or more p62 spots. The cytoplasmic area of 300 p62-positive and 300 p62-negative myocytes randomly selected from 3 animals in each group was measured. A total of 9 sections from 3 animals in each group were used to determine the number of p62-positive myocytes.

(8) Western blot analysis

For Western blot analysis, the sciatic nerve and muscle were largely dissected into small pieces and then tissue lysates were prepared using TissueLyser LT (Qiagen) in a modified RIPA buffer containing 1% Triton X-100 in Tris-EDTA solution. . The RIPA lysate was centrifuged at 9000 g at 4° C. for 10 minutes and the supernatant was collected. Proteins (10-35 μg) were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Amersham Biosciences). After blocking with Tris buffered saline (TBST; pH. 7.2) containing Tween-20 containing 5% skim milk powder for 1 hour at room temperature, the membrane was incubated with primary antibody (1:500 in TBST containing 1% skim milk powder). -2000) overnight at 4°C. After washing three times with TBST, the membrane was incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Chemiluminescent reactions were performed using the ECL Western Blotting Detection System (GE Healthcare). Images were then detected using Luminogragh 3 and quantified with CS Analyzer 4 (ATTO). Quantification was performed with a density analyzer in CS Analyzer 4, and values were obtained from three independent experiments.

(9) Statistical method

Results were expressed as median ± standard error of the mean (SEM). ELISA data were analyzed using one-way analysis of variance followed by Kruskal-Wallis test and Sidak's multiple comparison test using GraphPad Prism version 9 (GraphPad Software Inc., La Jolla, CA). Correlation was evaluated using the Spearman and Pearson correlation coefficient, and the comparison of plasma p62 concentrations between the two groups was analyzed by the Mann-Whitney U test. Logistic regression and receiver operating characteristic (ROC) curve analysis were used to evaluate the diagnostic performance of plasma p62 in CMT patients.

2. Experimental results

(1) Patient demographic details

A total of 138 CMT patients and 55 normal controls were enrolled. Table 1 summarizes the baseline demographic and clinical characteristics of patients with CMT, including duration of disease, plasma p62 level, and clinical score. There was no significant difference in gender or age between the CMT group and the normal control group. The CMT group consisted of 19 different subtypes of CMT, with CMT1A (n = 70) being the most common, followed by CMTX1 (n = 25) and CMT2A (n = 11). Among the CMT patients, the demyelinating type (CMT1 and CMT4) was the most common with 64.5% (n=89), the axonal type (CMT2) with 14.5% (n=20), and the intermediate type (CMTX1, CMTDI and Int-CMT) with 21.0%. (n = 29) followed.

(2) Diagnosis of high plasma p62 level in CMT patients

According to the ELISA results, the median plasma p62 concentration of HC was 1978 pg/ml, and the median plasma p62 concentration of CMT patients was 2354 pg/ml (p < 0.001; Table 1 and Fig. 1A). High plasma p62 levels were observed in all subtypes of CMT (demyelinated, axonal, or intermediate types), and plasma p62 concentrations were higher in demyelinated and axonal types of CMT than in HC, the most common genetic subtype. It was significantly increased in CMT1A.

When the ROC curve was plotted to determine how accurately assessing plasma p62 could predict CMT, the calculated “area under the curve (AUC)” was 0.644 ( FIG. 1D ). A plasma p62 concentration of 229.4 pg/ml or higher was found to be the best cut-off value to differentiate CMT and HC patients with 100% sensitivity and 100% specificity (FIG. 1E).

(3) Plasma p62 concentration reflecting disease severity and duration in CMT patients

Comparing the plasma p62 concentration levels among the three groups according to CMTNSv2, patients with CMTNSv2 ≥ 11 had higher median p62 concentrations than patients with CMTNSv2 ≤ 10 (p < 0.0001; Fig. 2A). Pearson's correlation analysis showed a significant correlation between plasma p62 levels and CMTNSv2 in CMT (r = 0.563, p < 0.0001, Figure 2B). Additionally, disease duration was a positive factor correlated with plasma p62 concentration in CMT (r = 0.281, p < 0.001, Figure 2C). According to this finding, age of onset was negatively correlated with plasma p62 concentration in CMT (r = 0.213, p < 0.05, Fig. 2D).

Next, clinical disability parameters and plasma p62 levels were compared in each subtype of CMT. In CMT1A patients, moderate or severe patients with CMTNSv2 ≥ 11 had higher plasma p62 levels than patients with mild disease (p < 0.0001), and there was a severity-related increase in plasma p62 concentration in Pearson's correlation analysis (r = 0.621, p < 0.0001, Fig. 3A, B). In CMT2A-affected patients, Pearson's correlation analysis showed a non-significant correlation between CMTNSv2 and plasma p62 concentrations (r = 0.602, p = 0.05, Fig. 3D), but in severely ill patients with CMTNSv2 ≥ 21 group showed higher plasma p62 levels than mild or moderate patients (p < 0.0001, Fig. 3C). CMTX1 patients showed a very high correlation score between plasma p62 concentration and CMTNSv2 (r = 0.751, p < 0.0001, Fig. 3E and F). Taken together, these findings suggest that plasma p62 concentrations represent disease severity in CMT.

(4) Increase of p62 expression in atrophic muscle and Schwann cells in C22 mouse, a mouse model of CMT1A

Since CMT is a chronic neuropathy that can cause secondary muscular atrophy with disease progression in all subtypes of CMT, in this example, we tested the expression of p62 in the gastrocnemius muscle of WT mice and C22 mice, an animal model for CMT1A, at 9 and 24 weeks after birth. expression was investigated. Western blot analysis showed that the level of p62 expression in muscle lysates increased with age in C22 mice compared to WT mice (Figures 4A and B). Immunofluorescence staining showed an increase in p62 speckle staining in C22 gastrocnemius myocytes (Fig. 4C). Comparison of the cytoplasmic areas of p62 p62-positive and -negative myocytes in the gastrocnemius of C22 mice at 24 weeks showed that the p62 speckle-positive myocytes were much smaller than the p62-negative myocytes, indicating that the p62-expressing myocytes may be atrophic myocytes (Fig. 4D). Finally, the average number of p62-positive atrophic cells was 54.3 per cross-sectional area of the gastrocnemius muscle in C22 mice, and there were no p62-positive atrophic cells in WT mice.

Muscle atrophy in C22 mice is known to occur due to neurological dysfunction. Therefore, as a result of testing whether p62 could induce muscle atrophy due to axotomy in wild-type C57BL/6 mice, it was shown that p62 expression was induced in the gastrocnemius muscle 4 weeks after sciatic nerve axotomy (Fig. 4E and F). Together, they indicate that atrophic muscle due to neurological dysfunction may provide a potential cause for plasma p62 elevation.

To determine p62 expression in the SC of C22 mice, we examined p62 protein expression in the sciatic nerve of C22 mice. Western blotting showed that p62 protein levels were elevated in the sciatic nerves of C22 mice at 24 weeks than in WT (Figures 5A and B). Immunofluorescence staining of p62 in the sciatic nerve showed punctate p62 staining in the perimyelin region of the myelinated SC of C22 mice at 9 weeks, and increased further at 24 weeks. However, punctate p62 immunostaining was barely detectable in the SCs of WT mice (Fig. 5C).

Claims (7)

delete delete delete A method for providing information for diagnosing Charcomaritus disease, which provides information that the individual has a higher probability of developing Charcomaritus disease compared to the control group when the concentration of p62 protein in serum or plasma isolated from the subject is higher than that of the control group.
The method according to claim 4, wherein the control group is a non-patient of Charcomaritus disease.
A method for providing information for diagnosing a Charcomaritus disease, wherein the information that the individual has a Charcomaritus disease is provided when the p62 protein concentration in serum or plasma isolated from the individual is higher than 2100 pg/ml.
If the p62 protein concentration in serum or plasma isolated from a patient with Charcomitus disease is higher than that of the control group, information for diagnosing Charcomitus disease, providing information that the patient is more likely to have severe Charcomitus disease compared to the control group How to provide.

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