WO2023214599A1 - Composition for diagnosis of charcot-marie-tooth disease and information provision method for diagnosis thereof - Google Patents

Composition for diagnosis of charcot-marie-tooth disease and information provision method for diagnosis thereof Download PDF

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WO2023214599A1
WO2023214599A1 PCT/KR2022/006439 KR2022006439W WO2023214599A1 WO 2023214599 A1 WO2023214599 A1 WO 2023214599A1 KR 2022006439 W KR2022006439 W KR 2022006439W WO 2023214599 A1 WO2023214599 A1 WO 2023214599A1
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marie
charcot
tooth disease
plasma
serum
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French (fr)
Korean (ko)
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박환태
조영래
김영희
윤별아
김종국
최병옥
남수현
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동아대학교 산학협력단
사회복지법인 삼성생명공익재단
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Definitions

  • the present invention relates to a composition for diagnosing Charcot-Marie-Tooth disease and a method for providing information for diagnosis.
  • Charcot-Marie-Tooth disease is a disease that causes progressive sensory impairment and muscle weakness, and is also called hereditary motor and sensory neuropathy.
  • Charcot-Marie-Tooth disease is mainly characterized by muscle weakness and atrophy that gradually progresses from the distal to the proximal lower extremities, including areflexia, distal sensory loss, pes cavus, and hearing loss. Deafness may also appear. The onset usually occurs around the teenage years, but rarely occurs after the age of 30.
  • There are currently over 120 known causative genes for Charcot-Marie-Tooth disease and although symptoms are caused by mutations in the same gene, the clinical phenotype and age of onset are very diverse, making diagnosis difficult in the absence of a family history. Nerve biopsy can be helpful in diagnosis, but it is an invasive test and is not easy to use for diagnosis.
  • next generation sequencing has been actively used in the diagnosis of diseases, and research is being conducted very actively to isolate causative genes and identify molecular biological mechanisms.
  • NGS next generation sequencing
  • patients without genetic diagnosis are also at risk of Charcot-Marie-Tooth disease. It amounts to about 30% of Additionally, clinical research on genetic treatments for Charcot-Marie-Tooth disease has recently been conducted, emphasizing the importance of accurate diagnosis of Charcot-Marie-Tooth disease.
  • Nerve biopsy is also an invasive test and has limitations in being used as a diagnostic test every time.
  • Biomarkers are indicators that can detect changes in the body using proteins, DNA, RNA, metabolites, etc. By using biomarkers, you can objectively measure the subject's normal or pathological state and degree of response to drugs. You can. Biomarkers are called the internal phenotype of a specific disease and can be used to find the cause of a specific disease or measure the risk of developing it.
  • TMPRSS5 neurofilament (NFL) or Schwann cells, which is detected at a higher rate in patients with Charcot-Marie-Tooth disease compared to non-patients.
  • a composition for diagnosing Charcot-Marie-Tooth disease comprising an agent for detecting the expression of p62 protein in serum or plasma.
  • composition for diagnosing Charcot-Marie-Tooth disease according to 1 above, wherein the Charcot-Marie-Tooth disease is selected from the group consisting of demyelinating type, axonal type, and intermediate type.
  • a kit for diagnosing Charcot-Marie-Tooth disease comprising the composition of any one of 1 or 2 above.
  • a method of providing information for diagnosing Charcot-Marie-Tooth disease which provides information that when the p62 protein concentration in serum or plasma isolated from an individual is higher than that of the control group, the individual has a higher probability of developing Charcot-Marie-Tooth disease compared to the control group.
  • a method of providing information for diagnosing Charcot-Marie-Tooth disease which provides information that the individual has developed Charcot-Marie-Tooth disease when the p62 protein concentration in serum or plasma isolated from the individual is higher than 229.4 pg/ml.
  • Charcot-Marie-Tooth disease diagnosis which provides information that if the p62 protein concentration in serum or plasma isolated from a patient with Charcot-Marie-Tooth disease is higher compared to the control group, the patient is more likely to have severe Charcot-Marie-Tooth disease compared to the control group How to provide information for .
  • the diagnostic composition and kit of the present invention can diagnose Charcot-Marie-Tooth disease in a non-invasive manner.
  • the method of the present invention is non-invasive and can provide information for diagnosing or determining the severity of Charcot-Marie-Tooth disease in a simple manner.
  • composition/method of the present invention can diagnose Charcot-Marie-Tooth disease with high accuracy or provide information necessary for diagnosis.
  • Figure 1 shows the results of an experiment on the level of p62 protein expression in patients with Charcot-Marie-Tooth disease compared to non-patients.
  • Figure 2 shows the results of an experiment on the level of p62 protein expression by Charcot-Marie-Tooth disease severity, onset period, and age.
  • Figure 3 shows the results of an experiment on the level of p62 protein expression according to severity in different subtypes according to gene type of Charcot-Marie-Tooth disease.
  • Figure 4 shows the results of an experiment on the level of p62 protein expression in muscle cells of C22 mice compared to WT mice.
  • Figure 5 shows the results of an experiment on the level of p62 protein expression in the sciatic nerve and Schwann cells of C22 mice compared to WT mice.
  • the present invention relates to a composition for diagnosing Charcot-Marie-Tooth disease.
  • composition for diagnosing Charcot-Marie-Tooth disease of the present invention includes an agent for detecting the expression of p62 protein in serum or plasma.
  • the present inventors designed the present invention based on the finding that the concentration of p62 protein in serum or plasma is positively correlated with the occurrence of Charcot-Marie-Tooth disease, and that the higher the concentration, the higher the severity of the disease.
  • composition of the present invention contains an agent for detecting the expression of p62 protein in serum or plasma, and can be used to measure the presence and concentration of p62 protein in serum or plasma, thereby diagnosing the occurrence of Charcot-Marie-Tooth disease or , can be used to diagnose its severity.
  • the p62 protein concentration in the serum or plasma of patients suspected of Charcot-Marie-Tooth disease with the concentration in the non-patient control group, if the p62 protein concentration in the serum or plasma of the patient suspected of Charcot-Marie-Tooth disease is higher in the non-patient control group, Compared to other cases, it can be diagnosed that the probability of developing Charcot-Marie-Tooth disease is higher.
  • the patient with the higher concentration of the protein can be diagnosed as more likely to have developed Charcot-Marie-Tooth disease or as having more advanced symptoms.
  • agent for detecting the expression of a protein in serum or plasma is not limited as long as it can detect it by interacting with p62, such as by binding to or reacting with the p62 protein in serum or plasma.
  • agent may be an antibody, aptamer, protein, peptide, nucleic acid, polymer, compound, or reagent.
  • Charcot-Marie-Tooth disease may be selected from the group consisting of demyelinating, axonal, and intermediate types. For example, it may be CMT1A in the demyelinating type, CMT2A in the axonal type, or CMTX1 in the intermediate type, but is not limited thereto.
  • the present invention relates to a kit for diagnosing Charcot-Marie-Tooth disease comprising the composition.
  • the kit not only includes a material that specifically binds to or reacts with the p62 protein, but may also include one or more additional compositions, components, devices, etc. suitable for an analysis method for measuring the protein expression level.
  • Analytical methods include, for example, BCA (Bicinchoninic acid) assay, Biuret reaction assay, Lowry test (Folin-Ciocalteau), Bradford assay, ultraviolet spectrophotometry, such as the Warburg-Christian method or Waddell method, and Micro-Kjeldahl assay. Alternatively, a combination of these may be used, but is not limited thereto, and protein analysis methods 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 chromogenic substrate solution for color development with the label, a washing solution, and an enzyme reaction stopping solution. It may include, and may be manufactured into a number of separate packaging or compartments containing the reagent components used, but is not limited thereto.
  • 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 antibodies.
  • the kit may be a kit characterized by including essential elements necessary for performing ELISA in order to implement various ELISA methods such as an ELISA kit and a sandwich ELISA kit.
  • These ELISA kits contain specific antibodies against the proteins.
  • the antibody has high specificity and affinity for the P62 protein and has little cross-reactivity to other proteins, and may be a monoclonal antibody, polyclonal antibody, or recombinant antibody.
  • ELISA kits may include antibodies specific for control proteins.
  • Other ELISA kits may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes and their substrates, or other substances capable of binding to antibodies. It may not be limited to this.
  • the kit may be a kit for implementing Western blot, immunoprecipitation analysis, complement fixation analysis, flow cytometry, or protein chip, and may further include additional components suitable for each analysis method.
  • Charcot-Marie-Tooth disease can be diagnosed by comparing the amount of antigen-antibody complex formation.
  • the present invention relates to a method of providing information for diagnosing Charcot-Marie-Tooth disease.
  • the method of the present invention provides information that when the p62 protein concentration in serum or plasma isolated from an individual is high compared to the control group, the individual has a higher probability of developing Charcot-Marie-Tooth disease compared to the control group.
  • the subject may be an animal suspected of having Charcot-Marie-Tooth disease, another animal for which information for Charcot-Marie-Tooth disease diagnosis is to be provided, and the animal may be a mammal, including a human.
  • the control group may be, for example, a non-patient with Charcot-Marie-Tooth disease, and the p62 protein concentration in the serum or plasma of the subject may be compared with the p62 protein concentration in the serum or plasma of the subject, or the mean value, median value, etc. of the group including them. It may be possible.
  • the p62 protein concentration in serum or plasma is higher than in other cases. Therefore, if the p62 protein concentration in the serum or plasma of the subject is higher than that of the control group, the probability of the subject developing Charcot-Marie-Tooth disease compared to the control group is higher. It can provide information that it is high.
  • the method of the present invention may further include measuring the p62 protein concentration in serum or plasma isolated from the subject and comparing it with the concentration in the control group.
  • the concentration measurement can be performed, for example, using the composition described above, and methods known in the art for measuring protein concentration can be used without limitation.
  • the present invention relates to a method of providing information for diagnosing Charcot-Marie-Tooth disease, which provides information that the individual has developed Charcot-Marie-Tooth disease when the p62 protein concentration in serum or plasma isolated from the individual is higher than 229.4 pg/ml. will be.
  • the p62 protein concentration value of 229.4 pg/ml in the serum or plasma is a cutoff value to distinguish between patients with Charcot-Marie-Tooth disease and the non-patient group. If the p62 protein concentration in the serum or plasma of an individual is higher than 229.4 pg/ml, the individual has Charcot-Marie-Tooth disease. It can provide information that maritus disease has occurred.
  • the method of the present invention may further include the step of measuring the p62 protein concentration in serum or plasma isolated from the subject, the description of which is as described above.
  • the present invention provides a Charcot-Marie-Tooth disease diagnosis that provides information that when the p62 protein concentration in serum or plasma isolated from a Charcot-Marie-Tooth disease patient is higher than that of the control group, the patient is more likely to have severe Charcot-Marie-Tooth disease compared to the control group. It is about how to provide information for.
  • Patients with Charcot-Marie-Tooth disease include animals with Charcot-Marie-Tooth disease or other animals seeking information for diagnosing Charcot-Marie-Tooth disease, and the animals may be mammals, including humans.
  • the control group may be, for example, a patient with Charcot-Marie-Tooth disease, and the p62 protein concentration in the serum or plasma of the subject is compared with the p62 protein concentration in the serum or plasma of the control patient, or the average value, median value, etc. of the control patient are compared. It may be possible.
  • the severity of Charcot-Marie-Tooth disease can be divided according to the degree of symptom expression due to the disease, and the higher the degree of symptom expression, the higher the severity of Charcot-Marie-Tooth disease.
  • These symptoms may include, for example, extremity muscle weakness and atrophy, neurogenic muscular atrophy, inhibition of autophagy in muscle, denervation-induced muscle loss, progressive muscle wasting, etc.
  • the severity of Charcot-Marie-Tooth disease can be measured using the CMT neuropathy score version 2 (CMTNSv2), with CMTNSv2 ⁇ 10 being mild, CMTNSv2 being 11-20 being moderate, and CMTNSv2 being ⁇ 21 being severe. It can be divided into three groups:
  • the Charcot-Marie-Tooth disease scoring system is not limited to this, and systems such as CMTPedS, CMT-FOM, or CMTInfS may be used.
  • the p62 protein concentration in serum or plasma is higher than in other cases, and the higher the degree of expression of the above symptoms, for example, the higher the CMTNSv2 value, the higher the p62 protein concentration in serum or plasma. If the p62 protein concentration in the patient's serum or plasma is higher than that of the control group, information may be provided that the patient is more likely to have severe Charcot-Marie-Tooth disease compared to the control group.
  • the method of the present invention may further include measuring the p62 protein concentration in serum or plasma isolated from the patient and comparing it with the concentration in the control group.
  • the concentration measurement can be performed, for example, using the composition described above, and methods known in the art for measuring protein concentration can be used without limitation.
  • the difference in p62 levels in the plasma of CMT patients and healthy controls (HC) was compared using ELISA using the p62-containing biomarker composition of the present invention.
  • plasma p62 levels in CMT patients and age-matched healthy controls were measured using ELISA to determine whether the plasma p62 concentration could be changed in CMT patients.
  • CMT patients were consecutively enrolled after approval from the Hereditary Neuropathy Clinic, Department of Neurology, Samsung Medical Center. Written consent was obtained from all participants according to a protocol approved by the Bioethics Committee (IRB) of Sungkyunkwan University Samsung Seoul Hospital (SMC, 2020-01-146). PMP22 replication causing CMT1A was determined by hexaplex microsatellite PCR on the chromosome 17p12 region. Other mutations were screened using whole exome sequencing and confirmed using Sanger sequencing. All CMT patients had confirmatory causative gene mutations (Table 1). Disease severity was measured using CMTNSv2. CMT patients were divided into three groups according to disease severity: mild (CMTNSv2 ⁇ 10), moderate (CMTNSv2, 11-20), and severe (CMTNSv2 ⁇ 21).
  • Demographic data, median p62 plasma concentration, CMTNSv2 and INCAT scores of CMT patients and HCs 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) N.A. Control 59 44.88 (1.069) 30/29 N.A. 1978 (1139-2522) N.A. N.A.
  • CMT4H One 26 0/1 20 4666 18 N.A.
  • CMT2EE One 31 0/1 18 3597 18 N.A.
  • CMT2F 2 37.5 (5.5) 0/2 16 (4) 899 (788-1011) 10.5 (10-11) N.A.
  • CMT2O One 45 0/1 20 3459 17 N.A.
  • CMT2S One 33 0/1 One 2282 10 N.A.
  • CMT2U One 63 1/0 5 5203 19 N.A.
  • HC plasma collection from HC
  • subjects who visited Dong-A University Hospital for health examination and matched in gender and age to the CMT group were enrolled.
  • Exclusion criteria included anemia, liver or kidney damage, or elevated fasting plasma glucose levels, which may contribute to peripheral neuropathy. Those with neurological symptoms or history of neurological disease were excluded.
  • Antibodies against p62, glyceraldehyde 3-phosphate dehydrogenase, and myelin basic protein (MBP) were purchased from Abcam (Cambridge, UK). Antibodies against neurofilament chain medium (NF-M) and ⁇ -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).
  • Blood samples were collected and processed within 1 hour. Blood was collected in SST tubes, centrifuged at 3,000 rpm for 10 minutes at 4°C, and plasma was aliquoted and stored at -80°C.
  • Measurement of plasma p62 was performed 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. For samples with an average p62 concentration of 2354 pg/ml, the average repeatability coefficient of variation was 7.31% and the inter-assay coefficient of variation was 8.78%. The limit of detection determined by the mean blank signal +3 SD for the p62 assay was 51.19 pg/ml. The lower limit of quantification (LLOQ), measured as the mean blank signal +10 SD, was 173.5 pg/ml. All samples in which p62 was detected under LLOQ were considered to have a concentration of 0.
  • LLOQ lower limit of quantification
  • C22 mice [B6; CBACa-Tg(PMP22)C22Clh/H] was purchased from Samsung Seoul Hospital.
  • the mouse model contains seven copies of the human Pmp22 gene, which causes demyelinating neuropathy.
  • Control C57BL/6 mice and C22 mice were perfusion-fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), and the sciatic nerve and gastrocnemius muscle were collected and cryoprotected in a 20% sucrose solution.
  • PBS phosphate-buffered saline
  • sections with a thickness of 10 ⁇ m were made using a cryocut machine and stored in the freezer until use. Slides were blocked with PBS containing 0.2% Triton X-100 and 5% bovine serum albumin for 1 hour at room temperature. Next, the slides were incubated with primary antibodies at 4°C overnight and then washed three times with PBS.
  • p62-positive myocytes were defined as myocytes with five 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 nine sections from three animals in each group were used to determine the number of p62-positive myocytes.
  • tissue lysates were prepared using a TissueLyser LT (Qiagen) in modified RIPA buffer containing 1% Triton X-100 in Tris-EDTA solution. .
  • the RIPA lysate was centrifuged at 9000 g for 10 min at 4°C and the supernatant was collected. Proteins (10-35 ⁇ g) were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Amersham Biosciences). After blocking for 1 hour at room temperature with Tris-buffered saline (TBST; pH.
  • the membrane was incubated with primary antibody (1:500) in TBST containing 1% nonfat dry milk. -2000) and cultured at 4°C overnight. After washing three times with TBST, the membrane was incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Chemiluminescence 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.
  • 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 assessed using Spearman and Pearson correlation coefficients, and comparisons of plasma p62 concentrations between the two groups were analyzed using 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.
  • ROC receiver operating characteristic
  • CMT1A 70
  • CMTX1 25
  • CMT2A 11
  • 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 Figure 1A).
  • High plasma p62 levels were observed in all subtypes of CMT (demyelinating, axonal, or intermediate). Plasma p62 levels were higher in demyelinating and axonal types of CMT than in HC, the most common genetic subtype. There was a significant increase in CMT1A.
  • CMT is a chronic neuropathy that can cause secondary muscle atrophy with disease progression in all subtypes of CMT
  • p62 was analyzed in the gastrocnemius muscle of WT mice and C22 mice, an animal model of CMT1A, at 9 and 24 weeks after birth. expression was investigated. Western blot analysis showed that p62 expression levels in muscle lysates increased with age in C22 mice compared to WT mice ( Figures 4A and B). Detection of p62 protein in C22 gastrocnemius muscle cells using immunofluorescence staining showed an increase in p62 spot staining in 24-week-old C22 mice, which is indicated by an arrow ( Figure 4C).
  • Muscle atrophy in C22 mice is known to be caused by neurological dysfunction. Therefore, we tested whether p62 can induce axotomy-induced muscle atrophy in wild-type C57BL/6 mice and showed that p62 expression was induced in the gastrocnemius muscle 4 weeks after sciatic nerve axotomy ( Fig. 4E and F ). Together, this indicates that atrophic muscles due to neurological dysfunction may provide a potential cause for elevated plasma p62.

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Abstract

The present invention relates to a composition for diagnosis of Charcot-Marie-Tooth disease and an information provision method for diagnosis thereof. The composition can be effectively used to diagnose Charcot-Marie-Tooth disease and determine the severity thereof, by detection of p62 protein in serum or plasma and measurement of the concentration thereof to compare same with a control group.

Description

샤르코마리투스 질환의 진단용 조성물 및 이의 진단을 위한 정보 제공 방법Composition for diagnosing Charcot-Marie-Tooth disease and method of providing information for the same
본 발명은 샤르코마리투스 질환 진단을 위한 조성물 및 진단을 위한 정보 제공 방법에 관한 것이다.The present invention relates to a composition for diagnosing Charcot-Marie-Tooth disease and a method for providing information for diagnosis.
샤르코마리투스 질환(Charcot-Marie-Tooth disease: CMT)은 진행하는 감각장애와 근위약이 나타나는 질환으로 유전성 운동 감각 신경질환(hereditary motor and sensory neuropathy)이라고도 한다. 샤르코마리투스 질환은 주로 하지의 말단에서부터 근위부로 서서히 진행되는 근력 약화와 위축을 특징으로 하며, 무반사증(areflexia), 말단의 감각 소실(distal sensory loss), 발모양의 기형화(pes cavus), 청력장애(deafness)가 나타나기도 한다. 발병은 10대 전후에 주로 일어나지만 드물게 30대 이후에도 일어난다. 샤르코마리투스 질환의 원인 유전자는 현재까지 120여 종류가 알려져 있고 동일한 유전자의 돌연변이에 의해 증상이 발생하더라도 임상적 표현형과 발병 연령은 매우 다양하여 가족력이 없는 경우 진단이 쉽지 않다. 신경 조직검사(nerve biopsy)가 진단에 도움이 될 수 있으나 침습적인 검사로 진단에 활용하기에 용이하지 않다.Charcot-Marie-Tooth disease (CMT) is a disease that causes progressive sensory impairment and muscle weakness, and is also called hereditary motor and sensory neuropathy. Charcot-Marie-Tooth disease is mainly characterized by muscle weakness and atrophy that gradually progresses from the distal to the proximal lower extremities, including areflexia, distal sensory loss, pes cavus, and hearing loss. Deafness may also appear. The onset usually occurs around the teenage years, but rarely occurs after the age of 30. There are currently over 120 known causative genes for Charcot-Marie-Tooth disease, and although symptoms are caused by mutations in the same gene, the clinical phenotype and age of onset are very diverse, making diagnosis difficult in the absence of a family history. Nerve biopsy can be helpful in diagnosis, but it is an invasive test and is not easy to use for diagnosis.
최근 차세대염기서열분석(next generation sequencing, NGS)이 질병의 진단에 활발히 활용되면서 원인 유전자를 분리하고 분자 생물학적 기전을 밝히는 연구가 매우 활발하게 진행되고 있으나, 유전자진단이 되지 않는 환자도 샤르코마리투스 질환의 약 30%에 이른다. 또한 최근 샤르코마리투스 질환의 유전치료제에 대한 임상연구가 진행되고 있어 샤르코마리투스 질환의 정확한 진단의 중요성이 강조되고 있다.Recently, next generation sequencing (NGS) has been actively used in the diagnosis of diseases, and research is being conducted very actively to isolate causative genes and identify molecular biological mechanisms. However, patients without genetic diagnosis are also at risk of Charcot-Marie-Tooth disease. It amounts to about 30% of Additionally, clinical research on genetic treatments for Charcot-Marie-Tooth disease has recently been conducted, emphasizing the importance of accurate diagnosis of Charcot-Marie-Tooth disease.
또한 샤르코마리투스 질환 진단 이후 환자 증상의 중증도를 평가하는 다양한 기술이 있으나, MRI나 전기전도 검사 등 대부분 고가이거나 매우 번거로운 방법이며, 혈청 또는 혈장을 이용하여 간단하지만 정확하게 평가하는 기술이 필요하다. 특히 최근 샤르코마리투스 질환의 맞춤형 치료 이후 중증도의 개선을 평가하는 방법을 개발하고자 하나 마땅히 보편적인 방법은 없는 실정이다.In addition, there are various techniques to evaluate the severity of a patient's symptoms after a diagnosis of Charcot-Marie-Tooth disease, but most of them, such as MRI or electrical conductivity tests, are expensive or very cumbersome, and a simple but accurate evaluation technique using serum or plasma is needed. In particular, we are currently trying to develop a method to evaluate the improvement in severity after customized treatment of Charcot-Marie-Tooth disease, but there is no universal method.
샤르코마리투스 질환의 임상적 표현형과 발병 연령이 매우 다양하며 약 30% 이상의 환자는 가족력이 없거나 차세대염기서열분석을 통해서도 정확한 진단을 받지 못한다. 신경 조직검사도 침습적인 검사로 매번 진단검사로 활용하기에는 한계가 있다.The clinical phenotype and age of onset of Charcot-Marie-Tooth disease are very diverse, and more than 30% of patients have no family history or cannot receive an accurate diagnosis even through next-generation sequencing. Nerve biopsy is also an invasive test and has limitations in being used as a diagnostic test every time.
이에 샤르코마리투스 질환을 정확하게 진단하고, 환자의 증상의 심각도를 판단할 수 잇는 신규 바이오마커에 대한 연구가 진행되고 있다. 바이오마커는 단백질이나 DNA, RNA, 대사물질 등을 이용해 몸 안의 변화를 알아낼 수 있는 지표를 의미하며, 바이오마커를 활용하면 대상체의 정상 또는 병리적인 상태, 약물에 대한 반응 정도 등을 객관적으로 측정할 수 있다. 바이오마커는 특정 질병의 내적 표현형이라고 불리며, 특정 질병의 원인을 찾거나 발병 위험도를 측정하는 데 이용할 수 있다.Accordingly, research is being conducted on new biomarkers that can accurately diagnose Charcot-Marie-Tooth disease and determine the severity of the patient's symptoms. Biomarkers are indicators that can detect changes in the body using proteins, DNA, RNA, metabolites, etc. By using biomarkers, you can objectively measure the subject's normal or pathological state and degree of response to drugs. You can. Biomarkers are called the internal phenotype of a specific disease and can be used to find the cause of a specific disease or measure the risk of developing it.
특히, 비환자와 비교하여 샤르코마리투스 질환 환자에게서 높게 검출되는 신경섬유실(neurofilament, NFL) 또는 슈반세포에 특이적으로 발현되는 유전자(TMPRSS5)를 바이오마커로 활용하기 위한 연구가 진행되고 있다.In particular, research is underway to use as a biomarker a gene (TMPRSS5) specifically expressed in neurofilament (NFL) or Schwann cells, which is detected at a higher rate in patients with Charcot-Marie-Tooth disease compared to non-patients.
그러나, 본 발명과 같은 비침습적으로 획득 가능한 혈청 또는 혈장을 활용한 단백질 바이오마커가 샤르코마리투스 질환 진단에 활용된 사례는 극히 드물다. 특히, 기존에 보고된 혈청 진단보조 단백질들은 비환자 대조군의 혈청에서도 존재하기 때문에 진단적 가치를 가지기가 어렵다. 따라서, 혈청 또는 혈장을 통한 간편하고 정확성이 높은 샤르코마리투스 질환 진단을 위한 단백질 바이오마커 개발이 요구된다.However, there are very few cases where protein biomarkers using serum or plasma that can be obtained non-invasively, such as the present invention, have been used to diagnose Charcot-Marie-Tooth disease. In particular, previously reported serum diagnostic aid proteins are difficult to have diagnostic value because they are also present in the serum of non-patient control subjects. Therefore, there is a need to develop protein biomarkers for simple and highly accurate diagnosis of Charcot-Marie-Tooth disease through serum or plasma.
1. 혈청 또는 혈장에서 p62 단백질의 발현을 검출하기 위한 제제를 포함하는, 샤르코마리투스 질환 진단용 조성물.1. A composition for diagnosing Charcot-Marie-Tooth disease, comprising an agent for detecting the expression of p62 protein in serum or plasma.
2. 위 1에 있어서, 샤르코마리투스 질환은 탈수초형(demyelinating type), 축삭형(axonal type), 및 중간형(intermediate type)으로 이루어진 군에서 선택되는 것인 샤르코마리투스 질환 진단용 조성물.2. The composition for diagnosing Charcot-Marie-Tooth disease according to 1 above, wherein the Charcot-Marie-Tooth disease is selected from the group consisting of demyelinating type, axonal type, and intermediate type.
3. 위 1 또는 2 중 어느 한 항의 조성물을 포함하는 샤르코마리투스 질환 진단용 키트.3. A kit for diagnosing Charcot-Marie-Tooth disease comprising the composition of any one of 1 or 2 above.
4. 개체로부터 분리된 혈청 또는 혈장 내의 p62 단백질 농도가 대조군 대비 높으면 상기 개체가 대조군 대비 샤르코마리투스 질환의 발병 확률이 높다는 정보를 제공하는, 샤르코마리투스 질환 진단을 위한 정보 제공 방법.4. A method of providing information for diagnosing Charcot-Marie-Tooth disease, which provides information that when the p62 protein concentration in serum or plasma isolated from an individual is higher than that of the control group, the individual has a higher probability of developing Charcot-Marie-Tooth disease compared to the control group.
5. 위 4에 있어서, 상기 대조군이 샤르코마리투스 질환 비환자인, 샤르코마리투스 질환 진단을 위한 정보 제공 방법.5. The method of providing information for diagnosing Charcot-Marie-Tooth disease in item 4 above, wherein the control group is a non-patient with Charcot-Marie-Tooth disease.
6. 개체로부터 분리된 혈청 또는 혈장 내 p62 단백질 농도가 229.4 pg/ml보다 높은 경우, 상기 개체가 샤르코마리투스 질환이 발병되었다는 정보를 제공하는, 샤르코마리투스 질환 진단을 위한 정보 제공 방법.6. A method of providing information for diagnosing Charcot-Marie-Tooth disease, which provides information that the individual has developed Charcot-Marie-Tooth disease when the p62 protein concentration in serum or plasma isolated from the individual is higher than 229.4 pg/ml.
7. 샤르코마리투스 질환 발병 환자로부터 분리된 혈청 또는 혈장 내 p62 단백질 농도가 대조군 대비 높으면, 상기 환자가 상기 대조군 대비 샤르코마리투스 질환이 중증일 가능성이 더 높다는 정보를 제공하는, 샤르코마리투스 질환 진단을 위한 정보 제공 방법.7. Charcot-Marie-Tooth disease diagnosis, which provides information that if the p62 protein concentration in serum or plasma isolated from a patient with Charcot-Marie-Tooth disease is higher compared to the control group, the patient is more likely to have severe Charcot-Marie-Tooth disease compared to the control group How to provide information for .
본 발명의 진단용 조성물 및 키트는 비침습적인 방법으로 샤르코마리투스 질환을 진단할 수 있다.The diagnostic composition and kit of the present invention can diagnose Charcot-Marie-Tooth disease in a non-invasive manner.
본 발명의 방법은 비침습적이며 간단한 방법으로 샤르코마리투스 질환의 진단 또는 중증도 파악을 위한 정보를 제공할 수 있다.The method of the present invention is non-invasive and can provide information for diagnosing or determining the severity of Charcot-Marie-Tooth disease in a simple manner.
본 발명의 조성물/방법은 높은 정확도로 샤르코마리투스 질환을 진단하거나 진단에 필요한 정보를 제공할 수 있다.The composition/method of the present invention can diagnose Charcot-Marie-Tooth disease with high accuracy or provide information necessary for diagnosis.
도 1은 샤르코마리투스 질환 비환자 대비 발병 환자에서의 p62 단백질 발현 정도 실험 결과이다.Figure 1 shows the results of an experiment on the level of p62 protein expression in patients with Charcot-Marie-Tooth disease compared to non-patients.
도 2는 샤르코마리투스 질환 중증도, 발병 기간 및 연령 별 p62 단백질 발현 정도 실험 결과이다.Figure 2 shows the results of an experiment on the level of p62 protein expression by Charcot-Marie-Tooth disease severity, onset period, and age.
도 3은 샤르코마리투스 질환의 유전자 종류에 따른 서로 다른 아형에서의 중증도 별 p62 단백질 발현 정도 실험 결과이다.Figure 3 shows the results of an experiment on the level of p62 protein expression according to severity in different subtypes according to gene type of Charcot-Marie-Tooth disease.
도 4는 WT 마우스 대비 C22 마우스의 근육세포에서의 p62 단백질 발현 정도 실험 결과이다.Figure 4 shows the results of an experiment on the level of p62 protein expression in muscle cells of C22 mice compared to WT mice.
도 5는 WT 마우스 대비 C22 마우스의 좌골 신경 및 슈반세포에서의 p62 단백질 발현 정도 실험 결과이다.Figure 5 shows the results of an experiment on the level of p62 protein expression in the sciatic nerve and Schwann cells of C22 mice compared to WT mice.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 샤르코마리투스 질환 진단용 조성물에 관한 것이다.The present invention relates to a composition for diagnosing Charcot-Marie-Tooth disease.
본 발명의 샤르코마리투스 질환 진단용 조성물은 혈청 또는 혈장에서 p62 단백질의 발현을 검출하기 위한 제제를 포함한다.The composition for diagnosing Charcot-Marie-Tooth disease of the present invention includes an agent for detecting the expression of p62 protein in serum or plasma.
본 발명자들은 혈청 또는 혈장에서의 p62 단백질 농도가 샤르코마리투스 질환 발병 여부와 양의 상관관계가 있고, 그 농도가 높을수록 질환의 중증도가 높음을 확인한 것에 착안하여 본 발명을 고안하였다.The present inventors designed the present invention based on the finding that the concentration of p62 protein in serum or plasma is positively correlated with the occurrence of Charcot-Marie-Tooth disease, and that the higher the concentration, the higher the severity of the disease.
본 발명의 조성물은 혈청 또는 혈장에서 p62 단백질의 발현을 검출하기 위한 제제를 포함함으로써, 혈청 또는 혈장에서 p62 단백질의 존부, 그 농도를 측정하는데 사용될 수 있고, 이에 샤르코마리투스 질환 발병 여부를 진단하거나, 그 중증도를 진단하는데에 사용될 수 있다.The composition of the present invention contains an agent for detecting the expression of p62 protein in serum or plasma, and can be used to measure the presence and concentration of p62 protein in serum or plasma, thereby diagnosing the occurrence of Charcot-Marie-Tooth disease or , can be used to diagnose its severity.
예를 들어, 샤르코마리투스 질환 의심 환자의 혈청 또는 혈장 내 p62 단백질 농도와 비환자 대조군에서의 농도를 비교하여 샤르코마리투스 질환 의심 환자의 혈청 또는 혈장 내 p62 단백질 농도가 더 높은 경우, 비환자 대조군 대비 샤르코마리투스 질환 발병 확률이 더 높다고 진단할 수 있다. 또는, 두 의심 환자의 혈청 또는 혈장 내 p62 단백질 농도를 비교하여 상기 단백질의 농도가 더 높은 환자의 경우, 샤르코마리투스 질환이 발병되었을 가능성이 더 높거나 증상이 더 많이 진행되었다고 진단할 수 있다.For example, by comparing the p62 protein concentration in the serum or plasma of patients suspected of Charcot-Marie-Tooth disease with the concentration in the non-patient control group, if the p62 protein concentration in the serum or plasma of the patient suspected of Charcot-Marie-Tooth disease is higher in the non-patient control group, Compared to other cases, it can be diagnosed that the probability of developing Charcot-Marie-Tooth disease is higher. Alternatively, by comparing the p62 protein concentration in the serum or plasma of two suspected patients, the patient with the higher concentration of the protein can be diagnosed as more likely to have developed Charcot-Marie-Tooth disease or as having more advanced symptoms.
혈청 또는 혈장에서 단백질의 발현을 검출하기 위한 제제는 혈청 또는 혈장 내의 p62 단백질과 결합하거나, 이와 반응하는 등으로 p62와 상호작용하여 이를 검출할 수 있는 것이라면 그 종류는 제한되지 않는다. 예를 들면, 항체, 압타머, 단백질, 펩타이드, 핵산, 고분자, 화합물 또는 시약 등일 수 있다. The type of agent for detecting the expression of a protein in serum or plasma is not limited as long as it can detect it by interacting with p62, such as 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.
샤르코마리투스 질환은 탈수초형, 축삭형 및 중간형으로 이루어진 군에서 선택된 것일 수 있다. 예를 들면, 탈수초형 중에서는 CMT1A, 축삭형 중에서는 CMT2A 또는 중간형 중에서는 CMTX1일 수 있으나 이에 제한되지 않는다.Charcot-Marie-Tooth disease may be selected from the group consisting of demyelinating, axonal, and intermediate types. For example, it may be CMT1A in the demyelinating type, CMT2A in the axonal type, or CMTX1 in the intermediate type, but is not limited thereto.
또한, 본 발명은 상기 조성물을 포함하는 샤르코마리투스 질환 진단용 키트에 관한 것이다.Additionally, the present invention relates to a kit for diagnosing Charcot-Marie-Tooth disease comprising the composition.
상기 키트는 p62 단백질에 특이적으로 결합하거나 반응하는 물질을 포함할 뿐만 아니라, 상기 단백질 발현량을 측정하는 분석법에 적합한 하나 이상의 추가 조성, 구성, 장치 등을 포함할 수 있다.The kit not only includes a material that specifically binds to or reacts with the p62 protein, but may also include one or more additional compositions, components, devices, etc. suitable for an analysis method for measuring the protein expression level.
분석법으로는 예를 들면, BCA(Bicinchoninic acid) 정량법, Biuret 반응에 의한 정량법, Lowry 시험(Folin-Ciocalteau), Bradford 정량법, 자외선 분광광도법, 예를 들어 Warburg-Christian법 또는 Waddell법, Micro-Kjeldahl 정량법 또는 이들의 조합을 이용할 수 있으나, 이에 제한되지 않으며, 당업계에 통상적으로 사용되는 단백질 분석법을 이용할 수 있다.Analytical methods include, for example, BCA (Bicinchoninic acid) assay, Biuret reaction assay, Lowry test (Folin-Ciocalteau), Bradford assay, ultraviolet spectrophotometry, such as the Warburg-Christian method or Waddell method, and Micro-Kjeldahl assay. Alternatively, a combination of these may be used, but is not limited thereto, and protein analysis methods commonly used in the art may be used.
상기 키트는 p62 단백질에 특이적으로 결합하는 항체, 기질과의 반응에 의해서 발색하는 표지체가 접합된 2차 항체 접합체(conjugate), 상기 표지체와 발색 반응할 발색 기질 용액, 세척액 및 효소반응 정지용액 등을 포함할 수 있으며, 사용되는 시약 성분을 포함하는 다수의 별도 패키징 또는 컴파트먼트로 제작될 수 있으나, 이에 제한되지 않는다.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 chromogenic substrate solution for color development with the label, a washing solution, and an enzyme reaction stopping solution. It may include, and may be manufactured into a number of separate packaging or compartments containing the reagent components used, but is not limited thereto.
또한, 상기 키트는 항체의 면역학적 검출을 위하여 기질, 적당한 완충용액, 검출 라벨로 표지된 2차 항체 및 발색 기질 등을 포함할 수 있으나, 이에 제한되지 않는다.Additionally, 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 antibodies.
구체적인 일례로, 상기 키트는 ELISA 키트, 샌드위치 ELISA 키트 등 다양한 ELISA 방법을 구현하기 위하여, ELISA를 수행하기 위해 필요한 필수 요소를 포함하는 것을 특징으로 하는 키트일 수 있다. 이러한 ELISA 키트는 상기 단백질에 대한 특이적인 항체를 포함한다. 항체는 P62 단백질에 대한 특이성 및 친화성이 높고 다른 단백질에 교차 반응성이 거의 없는 항체로, 모노클로날 항체, 폴리클로날 항체 또는 재조합 항체일 수 있다. 또한 ELISA 키트는 대조군 단백질에 특이적인 항체를 포함할 수 있다. 그 외 ELISA 키트는 결합된 항체를 검출할 수 있는 시약, 예를 들면, 표지된 2차 항체, 발색단(chromopores), 효소 및 그의 기질 또는 항체와 결합할 수 있는 다른 물질 등을 포함할 수 있으나, 이에 제한되지 않을 수 있다.As a specific example, the kit may be a kit characterized by including essential elements necessary for performing ELISA in order to implement various ELISA methods such as an ELISA kit and a sandwich ELISA kit. These ELISA kits contain specific antibodies against the proteins. The antibody has high specificity and affinity for the P62 protein and has little cross-reactivity to other proteins, and may be a monoclonal antibody, polyclonal antibody, or recombinant antibody. Additionally, ELISA kits may include antibodies specific for control proteins. Other ELISA kits may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes and their substrates, or other substances capable of binding to antibodies. It may not be limited to this.
이 외에도, 상기 키트는 웨스턴 블롯, 면역침전분석법, 보체 고정 분석법, 유세포분석, 또는 단백질 칩 등을 구현하기 위한 키트일 수 있으며, 각 분석 방법에 적합한 부가적인 구성을 추가로 포함할 수 있다. 이 분석 방법들을 통하여, 항원-항체 복합체 형성량을 비교함으로써 샤르코마리투스 질환을 진단할 수 있다.In addition, the kit may be a kit for implementing Western blot, immunoprecipitation analysis, complement fixation analysis, flow cytometry, or protein chip, and may further include additional components suitable for each analysis method. Through these analysis methods, Charcot-Marie-Tooth disease can be diagnosed by comparing the amount of antigen-antibody complex formation.
또한, 본 발명은 샤르코마리투스 질환 진단을 위한 정보 제공 방법에 관한 것이다.Additionally, the present invention relates to a method of providing information for diagnosing Charcot-Marie-Tooth disease.
본 발명의 방법은 개체로부터 분리된 혈청 또는 혈장 내 p62 단백질 농도가 대조군 대비 높으면, 상기 개체가 상기 대조군 대비 샤르코마리투스 질환 발병 확률이 높다는 정보를 제공한다.The method of the present invention provides information that when the p62 protein concentration in serum or plasma isolated from an individual is high compared to the control group, the individual has a higher probability of developing Charcot-Marie-Tooth disease compared to the control group.
개체는 샤르코마리투스 질환 발병이 의심되는 동물, 그 외 샤르코마리투스 질환 진단을 위한 정보를 제공받고자 하는 동물 등으로서, 상기 동물은 인간을 포함하는 포유류일 수 있다.The subject may be an animal suspected of having Charcot-Marie-Tooth disease, another animal for which information for Charcot-Marie-Tooth disease diagnosis is to be provided, and the animal may be a mammal, including a human.
대조군은 예를 들면, 샤르코마리투스 질환 비환자일 수 있고, 상기 개체의 혈청 또는 혈장 내 p62 단백질 농도를 이들의 혈청 또는 혈장 내 p62 단백질 농도와 비교하거나, 이들을 포함하는 그룹의 평균값, 중위값 등과 비교할 수도 있다.The control group may be, for example, a non-patient with Charcot-Marie-Tooth disease, and the p62 protein concentration in the serum or plasma of the subject may be compared with the p62 protein concentration in the serum or plasma of the subject, or the mean value, median value, etc. of the group including them. It may be possible.
샤르코마리투스 질환 환자는 혈청 또는 혈장 내 p62 단백질 농도가 그렇지 않은 경우에 비해 높으므로, 상기 개체의 혈청 또는 혈장 내 p62 단백질 농도가 대조군 대비 높으면, 상기 개체가 상기 대조군 대비 샤르코마리투스 질환 발병 확률이 높다는 정보를 제공할 수 있다.In patients with Charcot-Marie-Tooth disease, the p62 protein concentration in serum or plasma is higher than in other cases. Therefore, if the p62 protein concentration in the serum or plasma of the subject is higher than that of the control group, the probability of the subject developing Charcot-Marie-Tooth disease compared to the control group is higher. It can provide information that it is high.
본 발명의 방법은 개체로부터 분리된 혈청 또는 혈장 내 p62 단백질 농도를 측정하고 이를 대조군에서의 농도와 비교하는 단계를 더 포함할 수 있다.The method of the present invention may further include measuring the p62 protein concentration in serum or plasma isolated from the subject and comparing it with the concentration in the control group.
상기 농도 측정은 예를 들면, 전술한 조성물을 사용하여 수행될 수 있고, 단백질 농도 측정에 사용되는 당 분야에 공지된 방법이 제한 없이 사용될 수 있다.The concentration measurement can be performed, for example, using the composition described above, and methods known in the art for measuring protein concentration can be used without limitation.
상기 농도 비교 또한 당 분야에 공지된 방법이 제한 없이 적용될 수 있다.In addition to the concentration comparison, methods known in the art can be applied without limitation.
또한 본 발명은 개체로부터 분리된 혈청 또는 혈장 내 p62 단백질 농도가 229.4 pg/ml보다 높은 경우, 상기 개체가 샤르코마리투스 질환이 발병되었다는 정보를 제공하는 샤르코마리투스 질환 진단을 위한 정보 제공 방법에 관한 것이다.In addition, the present invention relates to a method of providing information for diagnosing Charcot-Marie-Tooth disease, which provides information that the individual has developed Charcot-Marie-Tooth disease when the p62 protein concentration in serum or plasma isolated from the individual is higher than 229.4 pg/ml. will be.
상기 혈청 또는 혈장 내 p62 단백질 농도값 229.4 pg/ml는 샤르코마리투스 질환 환자와 비환자군을 구분하는 컷오프 값으로서, 개체의 혈청 또는 혈장 내 p62 단백질 농도가 229.4 pg/ml보다 높은 경우 상기 개체가 샤르코마리투스 질환이 발병되었다는 정보를 제공할 수 있다.The p62 protein concentration value of 229.4 pg/ml in the serum or plasma is a cutoff value to distinguish between patients with Charcot-Marie-Tooth disease and the non-patient group. If the p62 protein concentration in the serum or plasma of an individual is higher than 229.4 pg/ml, the individual has Charcot-Marie-Tooth disease. It can provide information that maritus disease has occurred.
본 발명의 방법은 개체로부터 분리된 혈청 또는 혈장 내 p62 단백질 농도를 측정하는 단계를 더 포함할 수 있으며, 이에 대한 설명은 전술한 바와 같다.The method of the present invention may further include the step of measuring the p62 protein concentration in serum or plasma isolated from the subject, the description of which is as described above.
또한 본 발명은 샤르코마리투스 질환 환자로부터 분리된 혈청 또는 혈장 내 p62 단백질 농도가 대조군 대비 높으면, 상기 환자가 상기 대조군 대비 샤르코마리투스 질환이 중증일 가능성이 더 높다는 정보를 제공하는 샤르코마리투스 질환 진단을 위한 정보 제공 방법에 관한 것이다.In addition, the present invention provides a Charcot-Marie-Tooth disease diagnosis that provides information that when the p62 protein concentration in serum or plasma isolated from a Charcot-Marie-Tooth disease patient is higher than that of the control group, the patient is more likely to have severe Charcot-Marie-Tooth disease compared to the control group. It is about how to provide information for.
샤르코마리투스 질환 환자는 샤르코마리투스 질환이 발병된 동물, 그 외 샤르코마리투스 질환 진단을 위한 정보를 제공받고자 하는 동물 등으로서, 상기 동물은 인간을 포함하는 포유류일 수 있다.Patients with Charcot-Marie-Tooth disease include animals with Charcot-Marie-Tooth disease or other animals seeking information for diagnosing Charcot-Marie-Tooth disease, and the animals may be mammals, including humans.
대조군은 예를 들면, 샤르코마리투스 질환 환자일 수 있고, 상기 개체의 혈청 또는 혈장 내 p62 단백질 농도를 상기 대조군 환자의 혈청 또는 혈장 내 p62 단백질 농도와 비교하거나, 상기 대조군 환자들의 평균값, 중위값 등과 비교할 수도 있다.The control group may be, for example, a patient with Charcot-Marie-Tooth disease, and the p62 protein concentration in the serum or plasma of the subject is compared with the p62 protein concentration in the serum or plasma of the control patient, or the average value, median value, etc. of the control patient are compared. It may be possible.
샤르코마리투스 질환의 중증도는 질환으로 인한 증상의 발현 정도에 따라 나누어질 수 있고, 증상 발현 정도가 높을수록 샤르코마리투스 질환의 중증도가 높다고 할 수 있다. 상기 증상에는 예를 들면, 말단 근육 약화 및 위축, 신경성 근위축, 근육에서의 자가포식 억제, 탈신경 유발성 근육 손실, 진행성 근육 소모 등이 있을 수 있다.The severity of Charcot-Marie-Tooth disease can be divided according to the degree of symptom expression due to the disease, and the higher the degree of symptom expression, the higher the severity of Charcot-Marie-Tooth disease. These symptoms may include, for example, extremity muscle weakness and atrophy, neurogenic muscular atrophy, inhibition of autophagy in muscle, denervation-induced muscle loss, progressive muscle wasting, etc.
예를 들면, 샤르코마리투스 질환의 중증도는 CMT neuropathy score version 2(CMTNSv2)를 이용하여 측정될 수 있으며, CMTNSv2 ≤ 10 인 경우 경증, CMTNSv2 값이 11-20인 경우 중등도, CMTNSv2 ≥ 21인 경우 중증의 세 그룹으로 나누어질 수 있다. 샤르코마리투스 질환 점수화 체계는 이에 제한되는 것이 아니며, CMTPedS, CMT-FOM, 또는 CMTInfS 등의 체계가 이용될 수 있다.For example, the severity of Charcot-Marie-Tooth disease can be measured using the CMT neuropathy score version 2 (CMTNSv2), with CMTNSv2 ≤ 10 being mild, CMTNSv2 being 11-20 being moderate, and CMTNSv2 being ≥ 21 being severe. It can be divided into three groups: The Charcot-Marie-Tooth disease scoring system is not limited to this, and systems such as CMTPedS, CMT-FOM, or CMTInfS may be used.
샤르코마리투스 질환 환자는 혈청 또는 혈장 내 p62 단백질 농도가 그렇지 않은 경우에 비해 높고, 상기 증상의 발현 정도가 높을수록, 예를 들면 CMTNSv2 값이 클수록, 혈청 또는 혈장 내 p62 단백질 농도가 높아지므로, 상기 환자의 혈청 또는 혈장 내 p62 단백질 농도가 대조군 대비 높으면, 상기 환자가 상기 대조군 대비 샤르코마리투스 질환이 중증일 가능성이 높다는 정보를 제공할 수 있다.In patients with Charcot-Marie-Tooth disease, the p62 protein concentration in serum or plasma is higher than in other cases, and the higher the degree of expression of the above symptoms, for example, the higher the CMTNSv2 value, the higher the p62 protein concentration in serum or plasma. If the p62 protein concentration in the patient's serum or plasma is higher than that of the control group, information may be provided that the patient is more likely to have severe Charcot-Marie-Tooth disease compared to the control group.
본 발명의 방법은 환자로부터 분리된 혈청 또는 혈장 내 p62 단백질 농도를 측정하고 이를 대조군에서의 농도와 비교하는 단계를 더 포함할 수 있다.The method of the present invention may further include measuring the p62 protein concentration in serum or plasma isolated from the patient and comparing it with the concentration in the control group.
상기 농도 측정은 예를 들면, 전술한 조성물을 사용하여 수행될 수 있고, 단백질 농도 측정에 사용되는 당 분야에 공지된 방법이 제한 없이 사용될 수 있다.The concentration measurement can be performed, for example, using the composition described above, and methods known in the art for measuring protein concentration can be used without limitation.
상기 농도 비교 또한 당 분야에 공지된 방법이 제한 없이 적용될 수 있다.In addition to the concentration comparison, methods known in the art can be applied without limitation.
이하, 본 발명을 구체적으로 설명하기 위해 실시예를 들어 상세하게 설명하기로 한다.Hereinafter, the present invention will be described in detail with reference to examples.
실시예. ELISA로 측정한 혈장 p62 수준 차이로 정상대조군으로부터 CMT 환자 식별 및 중증도 별 CMT 환자 구분Example. Identification of CMT patients from normal controls and classification of CMT patients by severity based on differences in plasma p62 levels measured by ELISA
본 발명의 p62를 포함하는 바이오마커 조성물을 이용하여 ELISA로 CMT 환자와 건강한 대조군(Healthy Control, HC)의 혈장에서의 p62 수준 차이를 비교하였다. 본 실시예에서는 병리학적 상태에서의 p62의 분비를 고려하여 CMT 환자에서 혈장 p62 농도가 변경될 수 있는지 확인하고자 CMT 환자와 연령이 일치하는 건강한 대조군의 혈장 p62 수준 측정을 ELISA로 수행하였다. 또한, CMT 아형의 다양한 임상 매개변수와 혈장 p62 농도의 상관관계 조사뿐 아니라 CMT1A의 동물 모델을 사용하여 혈장 p62의 잠재적 원천을 식별하고자 하였다. 보다 상세한 실험 과정은 아래에 기술하였다.The difference in p62 levels in the plasma of CMT patients and healthy controls (HC) was compared using ELISA using the p62-containing biomarker composition of the present invention. In this example, considering the secretion of p62 in pathological conditions, plasma p62 levels in CMT patients and age-matched healthy controls were measured using ELISA to determine whether the plasma p62 concentration could be changed in CMT patients. In addition, we sought to identify potential sources of plasma p62 using an animal model of CMT1A, as well as to investigate the correlation of plasma p62 concentrations with various clinical parameters of CMT subtypes. The more detailed experimental process is described below.
1. 실험대상 및 방법1. Experimental subjects and methods
(1) 참가자 및 연구 설계(1) Participants and study design
2020년부터 2021년까지 삼성서울병원 신경과 유전 신경병증 클리닉에서 승인을 받은 후 138명의 CMT 환자를 연속적으로 등록하였다. 성균관대학교 삼성서울병원 생명윤리위원회(IRB)로부터 승인 받은 프로토콜에 따라 모든 참가자로부터 서면 동의를 받았다 (SMC, 2020-01-146). CMT1A를 유발하는 PMP22 복제는 염색체 17p12 영역에 대한 헥사플렉스 미세위성 PCR (hexaplex microsatellite PCR) 에 의해 결정되었다. 전체 엑솜 시퀀싱을 사용하여 다른 돌연변이를 스크리닝하고 생어 염기서열 분석(Sanger sequencing)을 이용하여 확인하였다. 모든 CMT 환자는 확증적 원인 유전자 돌연변이가 있었다 (표 1). 질병의 중증도는 CMTNSv2를 이용하여 측정하였다. CMT 환자는 질병의 중증도에 따라 경증 (CMTNSv2 ≤ 10), 중등도 (CMTNSv2, 11-20), 중증 (CMTNSv2 ≥ 21) 의 세 그룹으로 나뉘었다.From 2020 to 2021, 138 CMT patients were consecutively enrolled after approval from the Hereditary Neuropathy Clinic, Department of Neurology, Samsung Medical Center. Written consent was obtained from all participants according to a protocol approved by the Bioethics Committee (IRB) of Sungkyunkwan University Samsung Seoul Hospital (SMC, 2020-01-146). PMP22 replication causing CMT1A was determined by hexaplex microsatellite PCR on the chromosome 17p12 region. Other mutations were screened using whole exome sequencing and confirmed using Sanger sequencing. All CMT patients had confirmatory causative gene mutations (Table 1). Disease severity was measured using CMTNSv2. CMT patients were divided into three groups according to disease severity: mild (CMTNSv2 ≤ 10), moderate (CMTNSv2, 11-20), and severe (CMTNSv2 ≥ 21).
CMT 환자 및 HC의 인구통계학적 데이터, p62 혈장 농도 중앙값, CMTNSv2 및 INCAT 점수Demographic data, median p62 plasma concentration, CMTNSv2 and INCAT scores of CMT patients and HCs
Patient groupPatient group nn Age(y) (SEM)Age(y) (SEM) Sex, F/MSex,F/M Duration (SEM)Duration (SEM) p62, pg/mL (IQR)p62, pg/mL (IQR) CMTNSv2 (IQR)CMTNSv2 (IQR) INCAT (IQR)INCAT (IQR)
CMTCMT 138138 44.90 (1.174)44.90 (1.174) 58/8058/80 22.67 (1.372)22.67 (1.372) 2354 (1603-3622)2354 (1603-3622) 14.0 (9-18)14.0 (9-18) NAN.A.
Control Control 5959 44.88 (1.069)44.88 (1.069) 30/2930/29 NAN.A. 1978 (1139-2522)1978 (1139-2522) NAN.A. NAN.A.
탈수초형 CMT Demyelinating CMT
CMT1ACMT1A 7070 47.26 (1.544)47.26 (1.544) 34/3634/36 21.66 (2.101)21.66 (2.101) 2520 (1820-3958)2520 (1820-3958) 13 (8-18)13 (8-18) NAN.A.
CMT1BCMT1B 66 43.5 (6.776)43.5 (6.776) 3/33/3 31.67 (8.739)31.67 (8.739) 1957 (1486-2166)1957 (1486-2166) 16.5 (14.25-18.75)16.5 (14.25-18.75) NAN.A.
CMT1CCMT1C 1One 5656 1/01/0 1616 26982698 1616 NAN.A.
CMT1ECMT1E 88 39.38 (6.118)39.38 (6.118) 4/44/4 25.13 (5.761)25.13 (5.761) 1781 (1637-3555)1781 (1637-3555) 12.5 (8.25-19.75)12.5 (8.25-19.75) NAN.A.
CMT4B3CMT4B3 22 63 (2)63 (2) 2/02/0 55 (1)55 (1) 4218 (3756-4681)4218 (3756-4681) 2424 NAN.A.
CMT4FCMT4F 1One 4545 0/10/1 4545 42694269 3232 NAN.A.
CMT4HCMT4H 1One 2626 0/10/1 2020 46664666 1818 NAN.A.
축삭형 CMT Axial CMT
CMT2ACMT2A 11 11 46 (3.737)46 (3.737) 6/56/5 29.27 (3.231)29.27 (3.231) 1739 (1212-2544)1739 (1212-2544) 15 (10-20)15 (10-20) NAN.A.
CMT2EECMT2EE 1One 3131 0/10/1 1818 35973597 1818 NAN.A.
CMT2FCMT2F 22 37.5 (5.5)37.5 (5.5) 0/20/2 16 (4)16 (4) 899 (788-1011)899 (788-1011) 10.5 (10-11)10.5 (10-11) NAN.A.
CMT2OCMT2O 1One 4545 0/10/1 2020 34593459 1717 NAN.A.
CMT2SCMT2S 1One 3333 0/10/1 1One 22822282 1010 NAN.A.
CMT2UCMT2U 1One 6363 1/01/0 55 52035203 1919 NAN.A.
Other CMT2OtherCMT2 aa 33 44.0 (5.686)44.0 (5.686) 0/30/3 17 (6.11)17 (6.11) 3735 (403-3816)3735 (403-3816) 11 (10-18)11 (10-18) NAN.A.
중간형 CMTMedium CMT
CMTX1CMTX1 2525 39.76 (2.936)39.76 (2.936) 5/205/20 18.85 (2.153)18.85 (2.153) 2123 (1509-3395)2123 (1509-3395) 13 (9.5-16.5)13 (9.5-16.5) NAN.A.
CMTDIC CMTDIC 1One 5959 1/01/0 4242 60046004 1919 NAN.A.
CMTDIF CMTDIF 1One 2020 1/01/0 1414 381381 1313 NAN.A.
CMTDIGCMTDIG 1One 5252 0/10/1 3535 12301230 1414 NAN.A.
Int-CMTInt-CMT bb 1One 4949 0/10/1 2020 36953695 1818 NAN.A.
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 applicableAbbreviations: 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
aOther CMT2: axonal neuropathy CMT2 with KIF5A or RAB40B mutations a Other CMT2: axonal neuropathy CMT2 with KIF5A or RAB40B mutations
bInt-CMT: intermediate CMT neuropathy with DRP2 mutation b Int-CMT: intermediate CMT neuropathy with DRP2 mutation
HC로부터의 혈장 수집을 위해, 건강 검진을 위해 동아대학교 병원을 방문했고 CMT 그룹과 성별 및 연령이 일치하는 대상자를 등록하였다. 제외 기준에는 말초 신경병증에 영향을 미칠 수 있는 빈혈, 간 또는 신장 손상, 공복 혈장 포도당 수치 상승이 포함되었다. 신경학적 증상이 있거나 병력에 근거한 신경학적 질환이 있는 사람은 제외하였다.For plasma collection from HC, subjects who visited Dong-A University Hospital for health examination and matched in gender and age to the CMT group were enrolled. Exclusion criteria included anemia, liver or kidney damage, or elevated fasting plasma glucose levels, which may contribute to peripheral neuropathy. Those with neurological symptoms or history of neurological disease were excluded.
(2) 항체 및 시약(2) Antibodies and reagents
p62, glyceraldehyde 3-phosphate dehydrogenase 및 myelin basic protein (MBP)에 대한 항체는 Abcam (Cambridge, UK) 에서 구입하였다. 중간 크기의 신경섬유 사슬 (NF-M) 및 β-액틴에 대한 항체는 각각 Thermo Fisher Scientific (Waltham, MA, USA)과 Santa Cruz biotechnology (CA, USA) 에서 입수하였다. HRP(horseradish peroxidase)-연결 항토끼 IgG는 Cell Signaling Lab(Beverly, MA, USA)에서 구입하였다. Alexa Fluor 488 또는 Cy3-접합 이차 항체는 Molecular probes (Carlsbad, CA, USA) 에서 구입하였다. 달리 명시되지 않는 한, 다른 모든 시약은 Sigma-Aldrich (St. Louis, MO, USA) 에서 구입했다.Antibodies against p62, glyceraldehyde 3-phosphate dehydrogenase, and myelin basic protein (MBP) were purchased from Abcam (Cambridge, UK). Antibodies against neurofilament chain medium (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) 혈액 샘플 수집 및 보관(3) Blood sample collection and storage
혈액 샘플을 채취하여 1시간 이내에 처리하였다. 혈액을 SST 튜브에 수집하고 4℃에서 10분 동안 3,000rpm으로 원심분리 한 다음 혈장을 분취하여 -80℃에서 보관했다.Blood samples were collected and processed within 1 hour. Blood was collected in SST tubes, centrifuged at 3,000 rpm for 10 minutes at 4°C, and plasma was aliquoted and stored at -80°C.
(4) 표준 프로토콜 승인, 등록 및 환자 동의(4) standard protocol approval, registration, and patient consent;
연구 프로토콜은 동아대학교 생명윤리위원회(No. HR-004-02), 동아대학교병원(No. 13-042), 인제대학교 부산백병원(No. 2015-01-271), 삼성서울병원(No. 2020-01-146)으로부터 승인받았다.The research protocol was approved by 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), and Samsung Seoul Hospital (No. 2020). -01-146).
(5) p62 측정(5) p62 measurement
혈장 p62의 측정은 시판되는 ELISA 키트 (LSbio, LS-F49427, Seattle, USA) 를 사용하여 수행했다. 모든 샘플은 제조업체의 지침에 따라 희석 없이 3회 분석되었다. 평균 p62 농도가 2354 pg/ml인 샘플의 평균 반복성 변동 계수는 7.31%였고 분석 간 변동 계수는 8.78%였다. p62 분석에 대한 평균 공백 신호 +3 SD로 결정된 검출 한계는 51.19 pg/ml였다. 평균 공백 신호 +10 SD로 측정된 정량 하한선(LLOQ)은 173.5 pg/ml였다. LLOQ 하에서 p62가 검출된 모든 샘플은 농도가 0인 것으로 간주하였다.Measurement of plasma p62 was performed 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. For samples with an average p62 concentration of 2354 pg/ml, the average repeatability coefficient of variation was 7.31% and the inter-assay coefficient of variation was 8.78%. The limit of detection determined by the mean blank signal +3 SD for the p62 assay was 51.19 pg/ml. The lower limit of quantification (LLOQ), measured as the mean blank signal +10 SD, was 173.5 pg/ml. All samples in which p62 was detected under LLOQ were considered to have a concentration of 0.
(6) 동물 실험(6) Animal testing
모든 동물 실험은 동아대학교 동물연구위원회에서 승인한 프로토콜 (No. DIACUC 21-8) 에 따라 수행되었다. C22 마우스 [B6; CBACa-Tg(PMP22)C22Clh/H] 는 삼성서울병원에서 구입하였다. 마우스 모델에는 탈수초성 신경병증을 유발하는 인간 Pmp22 유전자 사본 7개가 포함되어 있다.All animal experiments were performed according to protocols approved by the Dong-A University Animal Research Committee (No. DIACUC 21-8). C22 mice [B6; CBACa-Tg(PMP22)C22Clh/H] was purchased from Samsung Seoul Hospital. The mouse model contains seven copies of the human Pmp22 gene, which causes demyelinating neuropathy.
좌골 신경 손상의 경우, 성인 C57BL/6 마우스의 왼쪽 좌골 신경을 10% 케타민 염산염 (Sanofi-Ceva, Duesseldorf, Germany; 0.1 ml/100 g body weight) 및 Rompun (Bayer, Leverkusen, Germany; 0.05 ml/100 g body weight) 혼합물로 마취한 후 정교한 홍채 가위 (FST Inc, Foster City, CA) 로 경비골의 분기점 근위 5mm에서 축삭 절단했다 (FST Inc, Foster City, CA). 퇴행성 신경 분석을 위해 병변 부위로부터 1 mm 길이의 말단 절단단을 버리고 표시된 시점에서 다음 5 mm 길이의 말단 절단단을 수집했다.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), and axotomy was performed 5 mm proximal to the bifurcation of the tibial bone using fine iris scissors (FST Inc, Foster City, CA). For neurodegenerative analysis, a 1 mm long distal residual limb was discarded from the lesion site and the next 5 mm long distal residual limb was collected at the indicated time points.
(7) 면역형광(IF) 염색 및 근세포 영역 측정(7) Immunofluorescence (IF) staining and measurement of myocyte area
대조군 C57BL/6 마우스와 C22 마우스를 인산완충식염수 (PBS) 에 녹인 4% 파라포름알데히드 (paraformaldehyde) 로 관류고정하고, 좌골신경과 비복근을 채취하여 20% 자당 용액에 동결보호하였다. p62 발현 분석을 위해 냉동조직절편기 (cryocut) 을 사용하여 두께 10 μm의 단면을 만들고 사용할 때까지 냉동고에 보관했다. 슬라이드는 실온에서 1시간 동안 0.2% Triton X-100 및 5% 소 혈청 알부민을 함유하는 PBS로 차단되었다. 다음으로, 슬라이드를 1차 항체와 함께 4℃에서 밤새 배양한 후, PBS로 3회 세척했다. 이후, 슬라이드를 Cy3- 또는 Alexa 488-접합 2차 항체와 함께 실온에서 3시간 동안 배양하고, DAPI로 30분 동안 염색하였다. 광학 현미경 분석을 위해, Zeiss AxioImager 2 또는 Image Express 공초점 현미경 (Carl Zeiss, Goettingen, Germany) 이 있는 ApoTome 형광현미경으로 염색된 섹션을 검사했다.Control C57BL/6 mice and C22 mice were perfusion-fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), and the sciatic nerve and gastrocnemius muscle were collected and cryoprotected in a 20% sucrose solution. To analyze p62 expression, sections with a thickness of 10 μm were made using a cryocut machine and stored in the freezer until use. Slides were blocked with PBS containing 0.2% Triton X-100 and 5% bovine serum albumin for 1 hour at room temperature. Next, the slides were incubated with primary antibodies at 4°C overnight and then washed three times with PBS. Then, the slides were incubated with Cy3- or Alexa 488-conjugated secondary antibodies at room temperature for 3 hours and stained with DAPI for 30 minutes. For light microscopic analysis, stained sections were examined under an ApoTome fluorescence microscope with a Zeiss AxioImager 2 or Image Express confocal microscope (Carl Zeiss, Goettingen, Germany).
비복근에서 p62-양성 반점이 있거나 없는 근세포의 세포질 면적을 측정하기 위해, p62-양성 근세포는 5개 이상의 p62 점을 갖는 근세포로 정의하였다. 각 그룹의 3마리 동물로부터 무작위로 선택된 300개의 p62-양성 및 300개 p62-음성 근세포의 세포질 면적을 측정하였다. p62-양성 근세포의 수를 결정하기 위해 각 그룹의 3마리 동물에서 총 9개의 섹션이 사용되었다.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 five 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 nine sections from three animals in each group were used to determine the number of p62-positive myocytes.
(8) 웨스턴 블롯 (Western blot) 분석(8) Western blot analysis
웨스턴 블롯 분석을 위해 좌골 신경과 근육을 작은 조각으로 크게 해부한 다음, Tris-EDTA 용액에 1% Triton X-100을 포함하는 변형된 RIPA 완충액에서 TissueLyser LT (Qiagen) 를 사용하여 조직 용해물을 만들었다. RIPA 용해물을 4℃에서 9000 g로 10분 동안 원심분리하고 상층액을 수집했다. 단백질 (10-35 μg) 을 SDS-PAGE로 분리한 다음 니트로셀룰로오스 막 (Amersham Biosciences) 으로 옮겼다. 5% 탈지 분유를 포함하는 Tween-20이 포함된 Tris 완충 식염수 (TBST; pH. 7.2) 로 실온에서 1시간 동안 차단한 후, 막을 1% 탈지 분유를 포함하는 TBST에서 1차 항체 (1:500-2000) 와 함께 하룻밤 동안 4℃에서 배양했다. TBST로 3회 세척한 후, 막을 HRP-접합 이차 항체와 함께 실온에서 1시간 동안 배양하였다. ECL 웨스턴 블롯팅 검출 시스템 (GE Healthcare) 을 사용하여 화학발광 반응을 수행하였다. 그런 다음 Luminogragh 3을 사용하여 이미지를 감지하고, CS Analyzer 4 (ATTO) 로 정량화했다. 정량화는 CS Analyzer 4의 밀도 분석기로 수행되었으며, 값은 3개의 독립적인 실험에서 얻어졌다.For Western blot analysis, the sciatic nerve and muscle were largely dissected into small pieces, and then tissue lysates were prepared using a TissueLyser LT (Qiagen) in modified RIPA buffer containing 1% Triton X-100 in Tris-EDTA solution. . The RIPA lysate was centrifuged at 9000 g for 10 min at 4°C and the supernatant was collected. Proteins (10-35 μg) were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Amersham Biosciences). After blocking for 1 hour at room temperature with Tris-buffered saline (TBST; pH. 7.2) containing Tween-20 containing 5% nonfat dry milk, the membrane was incubated with primary antibody (1:500) in TBST containing 1% nonfat dry milk. -2000) and cultured at 4°C overnight. After washing three times with TBST, the membrane was incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Chemiluminescence 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) 통계적 방법(9) Statistical methods
결과는 중앙값±평균의 표준 오차(SEM)로 표현되었다. ELISA 데이터는 단방향 분산 분석에 이어 GraphPad Prism 버전 9 (GraphPad Software Inc., La Jolla, CA) 를 사용한 Kruskal-Wallis 테스트 및 Sidak의 다중 비교 테스트를 사용하여 분석되었다. Spearman과 Pearson 상관 계수를 사용하여 상관관계를 평가하고, 두 그룹 간의 혈장 p62 농도 비교는 Mann-Whitney U 테스트로 분석했다. 로지스틱 회귀 및 수신자 작동 특성(ROC) 곡선 분석을 사용하여 CMT 환자에서 혈장 p62의 진단 성능을 평가했다.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 assessed using Spearman and Pearson correlation coefficients, and comparisons of plasma p62 concentrations between the two groups were analyzed using 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. 실험결과2. Experiment results
(1) 환자의 인구통계학적 세부정보(1) Patient demographic details
총 138명의 CMT 환자 및 55명의 정상대조군이 등록되었다. 표 1은 CMT 환자의 질병 기간, 혈장 p62 수준, 임상 점수를 포함한 기준 인구 통계 및 임상 특징을 요약한다. CMT 그룹과 정상대조군 그룹 사이에 성별이나 연령에는 유의한 차이가 없었다. CMT 그룹은 CMT의 19가지 다른 아형으로 이루어지는데, CMT1A (n = 70) 가 가장 흔했고, CMTX1 (n = 25) 과 CMT2A (n = 11) 가 그 뒤를 이었다. CMT 환자 중 탈수초형 (CMT1 및 CMT4) 이 64.5% (n=89) 로 가장 많았고, 축삭형 (CMT2)이 14.5% (n=20), 중간형 (CMTX1, CMTDI 및 Int-CMT)이 21.0% (n = 29) 로 그 뒤를 이었다.A total of 138 CMT patients and 55 normal controls were enrolled. Table 1 summarizes the baseline demographic and clinical characteristics of CMT patients, including disease duration, plasma p62 levels, and clinical scores. There was no significant difference in gender or age between the CMT group and the normal control group. The CMT group consists of 19 different subtypes of CMT, with CMT1A (n = 70) being the most common, followed by CMTX1 (n = 25) and CMT2A (n = 11). Among CMT patients, demyelinating type (CMT1 and CMT4) was the most common at 64.5% (n=89), axonal type (CMT2) at 14.5% (n=20), and intermediate type (CMTX1, CMTDI, and Int-CMT) at 21.0%. This was followed by (n = 29).
(2) CMT 환자에 대한 높은 혈장 p62 수준 진단(2) Diagnosis of high plasma p62 levels in CMT patients
ELISA 결과에 따르면 HC의 혈장 p62 농도는 중앙값 1978 pg/ml이었고, CMT 환자의 혈장 p62 농도는중앙값 2354 pg/ml이었다 (p < 0.001; 표 1 및 도 1A). 혈장의 높은 p62 수준은 CMT의 모든 아형 (탈수초형, 축삭형 또는 중간형) 에서 관찰되었으며, 혈장 p62 농도는 HC에서보다 CMT의 탈수초형, 축삭형에서 높게 나타났으며, 가장 흔한 유전적 아형인 CMT1A에서 유의성 있게 높게 증가했다.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 Figure 1A). High plasma p62 levels were observed in all subtypes of CMT (demyelinating, axonal, or intermediate). Plasma p62 levels were higher in demyelinating and axonal types of CMT than in HC, the most common genetic subtype. There was a significant increase in CMT1A.
혈장 p62를 평가하여 CMT를 얼마나 정확하게 예측할 수 있는지 결정하기 위해 ROC 곡선을 플로팅했을 때, 계산된 "곡선 아래 면적(AUC)"은 0.644이었다 (도 1D). 229.4 pg/ml 이상의 혈장 p62 농도는 100% 민감도 및 100% 특이도를 나타내는 CMT 및 HC 환자를 구별하는 가장 좋은 컷오프 값인 것으로 밝혀졌다 (도 1E).When the ROC curve was plotted to determine how accurately CMT could be predicted by assessing plasma p62, the calculated “area under the curve” (AUC) was 0.644 (Figure 1D). A plasma p62 concentration of 229.4 pg/ml or higher was found to be the best cutoff value to distinguish between CMT and HC patients, showing 100% sensitivity and 100% specificity (Figure 1E).
(3) CMT 환자의 질병 중증도 및 기간을 반영한 혈장 p62 농도(3) Plasma p62 concentration reflecting disease severity and duration in CMT patients
CMTNSv2에 따라 세 그룹 간의 혈장 p62 농도 수준을 비교했을 때, CMTNSv2 ≥ 11인 환자는 CMTNSv2 ≤ 10인 환자에 비해 p62 농도 중앙값이 더 높았다(p < 0.0001; 도 2A). 피어슨 상관관계 분석은 CMT에서 혈장 p62 수준과 CMTNSv2 사이에 유의한 상관 관계가 있음을 보여주었다(r = 0.563, p < 0.0001, 도 2B). 또한, 질병 기간은 CMT에서 혈장 p62 농도와 상관된 양의 인자였다(r = 0.281, p < 0.001, 도 2C). 이 발견에 따르면, 발병 연령은 CMT에서 혈장 p62 농도와 음의 상관관계가 있었다(r = 0.213, p < 0.05, 도 2D).When comparing plasma p62 concentration levels between the three groups according to CMTNSv2, patients with CMTNSv2 ≥ 11 had higher median p62 concentration compared to patients with CMTNSv2 ≤ 10 (p < 0.0001; Figure 2A). Pearson correlation analysis showed that there was 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, Figure 2D).
다음으로 CMT의 각 아형에서 임상 장애 매개변수와 혈장 p62 농도를 비교했다. CMT1A 환자에서 CMTNSv2 ≥ 11인 중등도 또는 중증 환자군은 경증 환자보다 혈장 p62 수치가 더 높았고 (p < 0.0001), 피어슨 상관관계 분석에서 중증도 관련 혈장 p62 농도 상승이 있었다 (r = 0.621, p < 0.0001, 도 3A, B). CMT2A에 영향을 받은 환자에서, 피어슨 상관관계 분석은 CMTNSv2와 혈장 p62 농도 사이에 유의하지 않은 상관관계가 있음을 보여주었지만 (r = 0.602, p = 0.05, 도 3D), CMTNSv2 ≥ 21인 중증 환자 그룹은 경증 또는 중등도 환자보다 높은 혈장 p62 수준을 보여줬다 (p < 0.0001, 도 3C). CMTX1 환자는 혈장 p62 농도와 CMTNSv2 사이에 매우 높은 상관 점수를 나타내었다 (r = 0.751, p < 0.0001, 도 3E 및 F). 종합하면, 혈장 p62 농도가 CMT에서 질병 중증도를 나타낸다는 것을 시사한다.Next, we compared clinical disability parameters and plasma p62 concentrations in each subtype of CMT. In CMT1A patients, the moderate or severe patient group with CMTNSv2 ≥ 11 had higher plasma p62 levels than mild patients (p < 0.0001), and there was a severity-related elevation of plasma p62 concentration in Pearson correlation analysis (r = 0.621, p < 0.0001, Fig. 3A, B). In patients affected by CMT2A, Pearson correlation analysis showed a non-significant correlation between CMTNSv2 and plasma p62 concentrations (r = 0.602, p = 0.05, Figure 3D), but only in critically ill patients with CMTNSv2 ≥ 21. group showed higher plasma p62 levels than mild or moderate patients (p < 0.0001, Figure 3C). CMTX1 patients showed a very high correlation score between plasma p62 concentration and CMTNSv2 (r = 0.751, p < 0.0001, Figure 3E and F). Taken together, this suggests that plasma p62 concentration is indicative of disease severity in CMT.
(4) CMT1A의 마우스 모델인 C22 마우스에서의 위축성 근육 및 슈반 세포 (Schwann Cell) 에서 p62 발현의 증가(4) Increase in p62 expression in atrophic muscles and Schwann cells in C22 mice, a mouse model of CMT1A
CMT는 CMT의 모든 아형에서 질병 진행과 함께 이차성 근위축을 유발할 수 있는 만성 신경병증이므로, 본 실시예에서는 출생 후 9주 및 24주에 WT 마우스 및 CMT1A의 동물 모델인 C22 마우스의 비복근에서 p62의 발현을 조사했다. 웨스턴 블롯 분석은 근육 용해물의 p62 발현 수준이 WT 마우스에 비해 C22 마우스에서 나이가 증가함에 따라 증가한다는 것을 보여주었다 (도 4A 및 B). 면역형광염색으로 C22 비복근 근세포에서 p62 단백질을 검출한 결과 24주의 C22 마우스에서 p62 반점 염색의 증가를 보여주었고, 이를 화살표로 표시하였다 (도 4C). 24주에 C22 생쥐의 비복근에서 p62 반점 양성 및 음성 근세포의 세포질 영역을 비교한 결과, p62 반점 양성 근세포가 p62 음성 근세포보다 훨씬 작아서 p62 발현 근세포가 위축성 근세포일 수 있음을 나타냈다 (도 4D). 마지막으로, p62 반점 양성 위축 세포의 평균 수는 C22 마우스에서 비복근의 단면적당 54.3개였으며, WT 마우스에서는 p62 반점 양성 위축 세포가 없었다.Since CMT is a chronic neuropathy that can cause secondary muscle atrophy with disease progression in all subtypes of CMT, in this example, p62 was analyzed in the gastrocnemius muscle of WT mice and C22 mice, an animal model of CMT1A, at 9 and 24 weeks after birth. expression was investigated. Western blot analysis showed that p62 expression levels in muscle lysates increased with age in C22 mice compared to WT mice (Figures 4A and B). Detection of p62 protein in C22 gastrocnemius muscle cells using immunofluorescence staining showed an increase in p62 spot staining in 24-week-old C22 mice, which is indicated by an arrow (Figure 4C). Comparison of the cytoplasmic areas of p62 speckle-positive and -negative myocytes in the gastrocnemius muscle of C22 mice at 24 weeks showed that p62 speckle-positive myocytes were much smaller than p62-negative myocytes, indicating that p62-expressing myocytes may be atrophic myocytes (Figure 4D). Finally, the average number of p62 spot-positive atrophic cells was 54.3 per cross-sectional area of the gastrocnemius muscle in C22 mice, while there were no p62 spot-positive atrophic cells in WT mice.
C22 마우스의 근육 위축은 신경 기능 장애로 인해 발생하는 것으로 알려져 있다. 따라서 p62가 야생형 C57BL/6 마우스에서 축삭 절제술로 인한 근육 위축을 유도할 수 있는지 여부를 테스트한 결과, 좌골신경 축삭절단 4주 후 비복근에서 p62 발현을 유도함을 보여주었다 (도 4E 및 F). 이와 함께, 신경 기능 장애로 인한 위축성 근육이 혈장 p62 상승의 잠재적인 원인을 제공할 수 있음을 나타낸다.Muscle atrophy in C22 mice is known to be caused by neurological dysfunction. Therefore, we tested whether p62 can induce axotomy-induced muscle atrophy in wild-type C57BL/6 mice and showed that p62 expression was induced in the gastrocnemius muscle 4 weeks after sciatic nerve axotomy ( Fig. 4E and F ). Together, this indicates that atrophic muscles due to neurological dysfunction may provide a potential cause for elevated plasma p62.
C22 마우스의 SC에서 p62 발현을 결정하기 위해, C22 마우스의 좌골 신경에서 p62 단백질 발현을 조사했다. 웨스턴 블롯팅은 24주에 C22 마우스의 좌골 신경에서 p62 단백질 수준이 WT에서보다 상승했음을 보여주었다 (도 5A 및 B). 좌골 신경을 ORO staining으로 염색한 후, 면역형광염색으로 p62의 수준을 측정하였다. 9주차에 C22 마우스의 수초화 SC의 수초 주위 영역에서 점상 p62 염색이 관찰되었고, 24주차에 더 증가하는 것으로 나타났다. 그러나 점상 p62 면역염색은 WT 마우스의 SC에서 거의 검출되지 않았다 (도 5C).To determine p62 expression in the SC of C22 mice, p62 protein expression was examined in the sciatic nerve of C22 mice. Western blotting showed that p62 protein levels in the sciatic nerves of C22 mice were elevated compared to those in WT at 24 weeks ( Fig. 5A and B ). After staining the sciatic nerve with ORO staining, the level of p62 was measured using immunofluorescence staining. At 9 weeks, punctate p62 staining was observed in the perimyelinating area of myelinated SC of C22 mice, and appeared to further increase at 24 weeks. However, punctate p62 immunostaining was barely detectable in the SCs of WT mice (Figure 5C).

Claims (7)

  1. 혈청 또는 혈장 내에서 p62 단백질의 발현을 검출하기 위한 제제를 포함하는, 샤르코마리투스 질환 진단용 조성물.A composition for diagnosing Charcot-Marie-Tooth disease, comprising an agent for detecting the expression of p62 protein in serum or plasma.
  2. 청구항 1에 있어서, 상기 샤르코마리투스 질환은 탈수초형, 축삭형, 및 중간형으로 이루어진 군에서 선택되는 것인 샤르코마리투스 질환 진단용 조성물.The composition for diagnosing Charcot-Marie-Tooth disease according to claim 1, wherein the Charcot-Marie-Tooth disease is selected from the group consisting of demyelinating type, axonal type, and intermediate type.
  3. 청구항 1 또는 2의 샤르코마리투스 질환 진단용 조성물을 포함하는 샤르코마리투스 질환 진단용 키트.A kit for diagnosing Charcot-Marie-Tooth disease, comprising the composition for diagnosing Charcot-Marie-Tooth disease of claim 1 or 2.
  4. 개체로부터 분리된 혈청 또는 혈장 내 p62 단백질 농도가 대조군 대비 높으면 상기 개체가 상기 대조군 대비 샤르코마리투스 질환의 발병 확률이 높다는 정보를 제공하는, 샤르코마리투스 질환 진단을 위한 정보 제공 방법.A method of providing information for diagnosing Charcot-Marie-Tooth disease, which provides information that when the p62 protein concentration in serum or plasma isolated from an individual is higher than that of the control group, the individual has a higher probability of developing Charcot-Marie-Tooth disease compared to the control group.
  5. 청구항 4에 있어서, 상기 대조군이 샤르코마리투스 질환 비환자인, 샤르코마리투스 질환 진단을 위한 정보 제공 방법.The method of claim 4, wherein the control group is a non-patient with Charcot-Marie-Tooth disease.
  6. 개체로부터 분리된 혈청 또는 혈장 내 p62 단백질 농도가 229.4 pg/ml보다 높은 경우, 상기 개체가 샤르코마리투스 질환이 발병되었다는 정보를 제공하는, 샤르코마리투스 질환 진단을 위한 정보 제공 방법.A method of providing information for diagnosing Charcot-Marie-Tooth disease, which provides information that the individual has developed Charcot-Marie-Tooth disease when the p62 protein concentration in the serum or plasma isolated from the individual is higher than 229.4 pg/ml.
  7. 샤르코마리투스 질환 환자로부터 분리된 혈청 또는 혈장 내 p62 단백질 농도가 대조군 대비 높으면, 상기 환자가 상기 대조군 대비 샤르코마리투스 질환이 중증일 가능성이 더 높다는 정보를 제공하는, 샤르코마리투스 질환 진단을 위한 정보 제공 방법.Information for diagnosing Charcot-Marie-Tooth disease, which provides information that if the p62 protein concentration in serum or plasma isolated from a Charcot-Marie-Tooth disease patient is higher compared to the control group, the patient is more likely to have severe Charcot-Marie-Tooth disease compared to the control group. How to provide.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5306616A (en) * 1991-06-06 1994-04-26 Baylor College Of Medicine Molecular diagnosis of autosomal dominant charcot-marie-tooth disease
WO2011061304A1 (en) * 2009-11-20 2011-05-26 Pharnext New diagnostic tools for charcot-marie-tooth disease
KR20180026990A (en) * 2016-09-05 2018-03-14 사회복지법인 삼성생명공익재단 Pharmaceutical Composition for treating Charcot Marie Tooth disease
KR101967880B1 (en) * 2017-08-09 2019-04-10 사회복지법인 삼성생명공익재단 Marker for diagnosing Charcot-Marie-Tooth disease and use thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6537095B2 (en) * 2014-10-24 2019-07-03 国立大学法人 筑波大学 Markers for predictive diagnosis of cancer recurrence and / or metastasis
KR101924269B1 (en) * 2016-12-30 2018-11-30 재단법인대구경북과학기술원 A method for preventing, attenuating or treating amyotrophic lateral sclerosis

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5306616A (en) * 1991-06-06 1994-04-26 Baylor College Of Medicine Molecular diagnosis of autosomal dominant charcot-marie-tooth disease
WO2011061304A1 (en) * 2009-11-20 2011-05-26 Pharnext New diagnostic tools for charcot-marie-tooth disease
KR20180026990A (en) * 2016-09-05 2018-03-14 사회복지법인 삼성생명공익재단 Pharmaceutical Composition for treating Charcot Marie Tooth disease
KR101967880B1 (en) * 2017-08-09 2019-04-10 사회복지법인 삼성생명공익재단 Marker for diagnosing Charcot-Marie-Tooth disease and use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
COLECCHIA DAVID, STASI MARIANGELA, LEONARDI MARGHERITA, MANGANELLI FIORE, NOLANO MARIA, VENEZIANI BIANCA MARIA, SANTORO LUCIO, ESK: "Alterations of autophagy in the peripheral neuropathy Charcot-Marie-Tooth type 2B", AUTOPHAGY, LANDES BIOSCIENCE, US, vol. 14, no. 6, 1 January 2018 (2018-01-01), US , pages 930 - 941, XP093106041, ISSN: 1554-8627, DOI: 10.1080/15548627.2017.1388475 *

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