METHOD AND KIT FOR DIAGNOSIS OF MUSCLE WEAKNESS-RELATED DISEASES USING BLOOD BIOMARKER

Abstract

The present invention relates to a composition and a kit for diagnosis of muscle weakness-related disease, which comprises agents for measuring the expression levels of gelsolin and tetranectin, and to a method of diagnosing muscle weakness-related disease by using the same. The composition, kit and method for diagnosis of muscle weakness-related disease according to the present invention make it possible to diagnose muscle weakness-related disease in an easy and rapid manner by molecular diagnosis, thereby systemically managing the muscle weakness-related disease while increasing therapeutic efficacy against the muscle weakness-related disease.

Claims

1-16. (canceled)

17. A kit for diagnosis of muscle weakness-related disease, which comprises: (a) an agent for measuring an expression level of gelsolin in a biological sample; and/or (b) an agent for measuring an expression level of tetranectin in the biological sample, wherein the (a) agent and/or the (b) agent contain a label which can be detected by any one selected from the group consisting of enzyme-linked immunosorbent (ELISA), radioimmunoassay (RIA), sandwich assay, Western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate color development, antigen-antibody aggregation, fluorescence activated cell sorter (FACS), mass spectrometry, MRM (multiple-reaction monitoring) assay, an assay employing a set of multiplexed, amine-specific, stable isotope reagents (iTRAQ, isobaric tags for relative and absolute quantitation), and protein chip assay.

18. The kit of claim 17, which further comprises (c) an agent for measuring any one or more proteins selected from the group consisting of macrophage migration inhibitory factor (MIF), interleukin-6 (IL-6), SPARC (secreted protein acidic and rich in cysteine), and insulin-like growth factor-1 (IGF-1) in the biological sample.

19. The kit of claim 17, wherein the muscle weakness-related disease is any one or more selected from the group consisting of sarcopenia, muscular atrophy, muscular dystrophy, cachexia, and acardiotrophy.

20. The kit of claim 17, wherein the (a) agent is a labeled antibody which specifically binds gelsolin, and/or the (b) agent is a labeled antibody which specifically binds to tetranectin.

21. The kit of claim 18, wherein the (a) agent is a labeled antibody which specifically binds gelsolin, and/or the (b) agent is a labeled antibody which specifically binds to tetranectin.

22. The kit of claim 19, wherein the (a) agent is a labeled antibody which specifically binds gelsolin, and/or the (b) agent is a labeled antibody which specifically binds to tetranectin.

23. The kit of claim 18, wherein the (c) agent is a labeled antibody which specifically binds to any one or more proteins selected from the group consisting of macrophage migration inhibitory factor (MIF), interleukin-6 (IL-6), SPARC (secreted protein acidic and rich in cysteine), and insulin-like growth factor-1 (IGF-1) in the biological sample.

24. The kit of claim 23, wherein the (c) agent comprises one or more selected from the group consisting of: (c-1) a labeled antibody specifically binding to macrophage migration inhibitory factor (MIF), (c-2) a labeled antibody specifically binding to interleukin-6 (IL-6), (c-3) a labeled antibody specifically binding to SPARC (secreted protein acidic and rich in cysteine), and (c-4) a labeled antibody specifically binding to insulin-like growth factor-1 (IGF-1).

25. A method for providing information for diagnosis of muscle weakness-related disease of a subject, the method comprising the steps of: (i) measuring expression levels of tetranectin and/or gelsolin proteins in a biological sample obtained from the subject; and (ii) determining that the subject has muscle weakness-related disease, when the expression levels of the tetranectin and/or gelsolin proteins are higher than reference expression levels of a control subject free of muscle weakness-related disease, wherein the (i) is carried out using (a) an agent for measuring the expression level of gelsolin and/or (b) an agent for measuring the expression level of tetranectin, wherein the (a) agent and/or the (b) agent contain a label which can be detected by any one selected from the group consisting of enzyme-linked immunosorbent (ELISA), radioimmunoassay (RIA), sandwich assay, Western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate color development, antigen-antibody aggregation, fluorescence activated cell sorter (FACS), mass spectrometry, MRM (multiple-reaction monitoring) assay, an assay employing a set of multiplexed, amine-specific, stable isotope reagents (iTRAQ, isobaric tags for relative and absolute quantitation), and protein chip assay.

26. The method of claim 25, wherein the biological sample in step (a) is any one selected from the group consisting of blood, serum, and plasma of the subject.

27. The method of claim 25, wherein, in (ii), the expression level of the gelsolin and/or the expression level of tetranectin of the subject are higher than about 10% of the reference levels, respectively, indicate that the subject has muscle weakness-related disease.

28. The method of claim 25, further comprises (iii) measuring expression level of any one or more proteins selected from the group consisting of macrophage migration inhibitory factor (MIF), interleukin-6 (IL-6), SPARC (secreted protein acidic and rich in cysteine) and insulin-like growth factor-1 (IGF-1), in the biological sample, wherein the (iii) is carried out using (c) an agent for measuring the expression level of the any one or more proteins selected from the group consisting of macrophage migration inhibitory factor (MIF), interleukin-6 (IL-6), SPARC (secreted protein acidic and rich in cysteine), and insulin-like growth factor-1 (IGF-1), and wherein the (c) agent contains a label which can be detected by any one selected from the group consisting of enzyme-linked immunosorbent (ELISA), radioimmunoassay (RIA), sandwich assay, Western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate color development, antigen-antibody aggregation, fluorescence activated cell sorter (FACS), mass spectrometry, MRM (multiple-reaction monitoring) assay, an assay employing a set of multiplexed, amine-specific, stable isotope reagents (iTRAQ, isobaric tags for relative and absolute quantitation), and protein chip assay.

29. The method of claim 28, further comprises (iv) determining that the subject has muscle weakness-related disease, when the expression levels of the any one or more proteins selected from the group consisting of macrophage migration inhibitory factor (MIF), interleukin-6 (IL-6), SPARC (secreted protein acidic and rich in cysteine), and insulin-like growth factor-1 (IGF-1) is higher than reference expression level of the control subject.

30. The method of claim 28, wherein, in (iv), the expression level of the gelsolin and the expression level of tetranectin of the subject are higher than about 10%/o of the reference levels, respectively, indicate that the subject has muscle weakness-related disease.

31. The method of claim 28, wherein the (i) and (iii) are carried out as a single test.

32. The method of claim 28, wherein the (c) agent comprises one or more selected from the group consisting of: (c-1) a labeled antibody specifically binding to macrophage migration inhibitory factor (MIF), (c-2) a labeled antibody specifically binding to interleukin-6 (IL-6), (c-3) a labeled antibody specifically binding to SPARC (secreted protein acidic and rich in cysteine), and (c-4) a labeled antibody specifically binding to insulin-like growth factor-1 (IGF-1).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0076] FIG. 1 shows the results of ELISA performed to measure serum MIF, IL-6, SPARC, IGF-1, gelsolin and tetranectin levels in a normal control group and a sarcopenia patient group.

[0077] FIG. 2 depicts receiver operating characteristics (ROC) graphs showing serum MIF, IL-6, SPARC, IGF-1, gelsolin and tetranectin levels in a normal control group and a sarcopenia patient group.

[0078] FIG. 3 shows the results of ROC curve analysis performed with a combination of two biomarkers (gelsolin and tetranectin) to confirm the classification of sarcopenia patients.

[0079] FIG. 4 shows the results of ROC curve analysis performed with a combination of three biomarkers (gelsolin, tetranectin and MIF) to confirm the classification of sarcopenia patients.

[0080] FIG. 5 shows the results of ROC curve analysis performed with a combination of three biomarkers (gelsolin, tetranectin and IL-6) to confirm the classification of sarcopenia patients.

[0081] FIG. 6 shows the results of ROC curve analysis performed with a combination of three biomarkers (gelsolin, tetranectin and SPARC) to confirm the classification of sarcopenia patients.

[0082] FIG. 7 shows the results of ROC curve analysis performed with a combination of three biomarkers (gelsolin, tetranectin and IGF-1) to confirm the classification of sarcopenia patients.

[0083] FIG. 8 shows the results of ROC curve analysis performed with a combination of six biomarkers (gelsolin, tetranectin, IL-6, SPARC, MIF and IGF-1) to confirm the classification of sarcopenia patients.

[0084] FIG. 9 shows the results of ELISA performed to measure serum gelsolin and tetranectin levels in muscular atrophy mouse models.

MODE FOR THE INVENTION

[0085] The advantages and features of the present invention, and the way of attaining them, will become apparent with reference to the examples described below. However, the present invention is not limited to the examples disclosed below and can be embodied in a variety of different forms; rather, these examples are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art. The scope of the present invention will be defined by the appended claims.

Example 1. Selection of Test Subjects and Measurement of Serum Protein Levels

[0086] In order to develop a method of diagnosing muscle weakness-related disease by use of blood biomarkers, among the elderly aged 60 or older, elderly persons with normal muscle mass and elderly persons with sarcopenia were selected as test subjects. The criteria for selection of sarcopenia were set as follows:

Appendicular skeletal muscle mass(ASM)ASM(kg)/height(m).sup.2:male<7.0 kg/m.sup.2,female<5.7 kg/m.sup.2

[0087] To measure serum proteins, serum protein levels were measured by an ELISA technique. Specifically, R&D systems Quantikine Elisa kits (Human IL-6, Cat # D6050; Human MIF, Cat # DMF00B; Human SPARC, Cat # DSP00; and Human IGF-1, DG100) and MyBioSource Elisa kits (Gelsolin, Cat # MBS7228324; Tetranectin, Cat # MBS762655) were used, and each serum protein level was measured using the protocol provided in each of the kits, thereby determining the serum levels of tetranectin, gelsolin, MIF (macrophage migration inhibitory factor), IL-6 (interleukin 6), SPARC (secreted protein acidic and rich in cysteine), and IGF-1 (Insulin-Like Growth Factor-1).

Example 2. Risk Score Calculation and Statistical Analysis

[0088] To compare the protein levels measured in Example 1, each protein level was log.sub.2 transformed and subjected to Logistic regression analysis.

[0089] The results are shown in FIG. 1.

[0090] As can be seen in FIG. 1, the serum levels of tetranectin, gelsolin, MIF, IL-6, SPARC and IGF-1 were all significantly different between the elderly persons with normal muscle mass and the elderly persons with sarcopenia. Specifically, the serum levels of tetranectin, gelsolin, MIF, IL-6 and SPARC were higher in the elderly persons with normal muscle mass than in the patient group with sarcopenia, and the serum level of IGF-1 was higher in the elderly persons with normal muscle mass.

[0091] Furthermore, to apply the measured proteins as multiple biomarkers, correction of a sarcopenia risk score according to each of the serum protein levels was performed. Specifically, the risk score for each protein was calculated by multiplying the linear regression coefficient corresponding to each protein in order to reduce the variable between the markers. The linear regression coefficient for each protein is shown in FIG. 1. The risk score of multiple markers was defined as the sum of the risk scores of the individual markers, and was calculated using the following Equation 1:

Risk score of multiple markers=logistic regression coefficient of molecule M.sub.ilog 2 transformed serum level of molecule M.sub.iEquation 1

[0092] In addition, each of the protein levels was expressed as a receiver operating characteristics (ROC) graph, and the results are shown in FIG. 2.

[0093] As can be seen in FIG. 2, tetranectin, gelsolin, MIF, IL-6, SPARC and IGF-1 all had high sensitivity, specificity and AUC values, suggesting that they can be used as single markers to diagnose sarcopenia.

[0094] In addition, all statistical analyses were performed using GraphPad Prism5 (GraphPad Software, Inc., USA) and R language environment (ver. 3.2.5). The difference between the control group and the test group was statistically analyzed by two tailed, unpaired Student's t-test, and sensitivity and specificity were calculated for combinations of the biomarkers, and AUC values were calculated using ROC curves. Furthermore, maximum value of the product of sensitivity and specificity was determined as cut-off and P value<0.05 was determined statistically significant.

Example 3. Analysis of Significance of Multiple Biomarkers for Diagnosis

[0095] (1) Analysis of Signification of Combination of Two Biomarkers (Gelsolin and Tetranectin) for Diagnosis

[0096] According to the method described in Example 2 above, the significance of a combination of two biomarkers (gelsolin and tetranectin) for diagnosis was analyzed. The results are shown in FIG. 3.

[0097] As shown in FIG. 3, the AUC value of the two-biomarker combination (gelsolin and tetranectin) in the elder persons with normal muscle mass and the elder persons with sarcopenia was 0.741, the maximum value of the product of sensitivity and specificity was 0.549, and the cut-off value was 2.512.

[0098] These results suggest that the two-biomarker combination (gelsolin and tetranectin) according to the present invention can diagnose sarcopenia with high accuracy.

[0099] (2) Analysis of Signification of Combination of Three Biomarkers (Including Gelsolin and Tetranectin) for Diagnosis

[0100] Analysis of Signification of Combination of Three Biomarkers (Gelsolin Tetranectin and MIF) for Diagnosis

[0101] According to the method described in Example 2 above, the significance of a combination of three biomarkers (gelsolin tetranectin and MIF) for diagnosis was analyzed. The results are shown in FIG. 4.

[0102] As shown in FIG. 4, the AUC value of the three-biomarker combination (gelsolin, tetranectin and MIF) in the elder persons with normal muscle mass and the elder persons with sarcopenia was 0.813, the maximum value of the product of sensitivity and specificity was 0.669, and the cut-off value was 3.886.

[0103] Analysis of Signification of Combination of Three Biomarkers (Gelsolin, Tetranectin and IL-6) for Diagnosis

[0104] According to the method described in Example 2 above, the significance of a combination of three biomarkers (gelsolin tetranectin and IL-6) for diagnosis was analyzed. The results are shown in FIG. 5.

[0105] As shown in FIG. 5, the AUC value of the three-biomarker combination (gelsolin, tetranectin and IL-6) in the elder persons with normal muscle mass and the elder persons with sarcopenia was 0.822, the maximum value of the product of sensitivity and specificity was 0.617, and the cut-off value was 2.746.

[0106] Analysis of Signification of Combination of Three Biomarkers (Gelsolin, Tetranectin and SPARC) for Diagnosis

[0107] According to the method described in Example 2 above, the significance of a combination of three biomarkers (gelsolin tetranectin and SPARC) for diagnosis was analyzed. The results are shown in FIG. 6.

[0108] As shown in FIG. 6, the AUC value of the three-biomarker combination (gelsolin, tetranectin and IL-6) in the elder persons with normal muscle mass and the elder persons with sarcopenia was 0.788, the maximum value of the product of sensitivity and specificity was 0.612, and the cut-off value was 3.287.

[0109] Analysis of Signification of Combination of Three Biomarkers (Gelsolin, Tetranectin and IGF-1) for Diagnosis

[0110] According to the method described in Example 2 above, the significance of a combination of three biomarkers (gelsolin tetranectin and SPARC) for diagnosis was analyzed. The results are shown in FIG. 7.

[0111] As shown in FIG. 7, the AUC value of the three-biomarker combination (gelsolin, tetranectin and IGF-1) in the elder persons with normal muscle mass and the elder persons with sarcopenia was 0.776, the maximum value of the product of sensitivity and specificity was 0.549, and the cut-off value was 1.822.

[0112] These results indicate that the three-biomarker combination including MIF, IL-6, SPARC or IGF-1 together with gelsolin and tetranectin shows a significantly increased AUC value compared to the two-biomarker combination of gelsolin and tetranectin.

[0113] (3) Analysis of Significance of Multiple Biomarkers (Gelsolin, Tetranectin, IL-6, MIF, SPARC and IGF-1) for Diagnosis

[0114] According to the method described in Example 2 above, the significance of multiple biomarkers (gelsolin, tetranectin IL-6, MIF, SPARC and IGF-1) for diagnosis was analyzed. The results are shown in FIG. 8.

[0115] As shown in FIG. 8, the total AUC value of gelsolin, tetranectin IL-6, MIF, SPARC and IGF-1 in the elder persons with normal muscle mass and the elder persons with sarcopenia was 0.877, which was the highest AUC value. In addition, the maximum value of the product of sensitivity and specificity was 0.668, and the cut-off value was 3.946.

[0116] From these results, it was found that the multiple biomarkers including gelsolin and tetranectin according to the present invention all had high sensitivity, specificity and AUC values, suggesting that they can be used as diagnostic biomarkers to detect sarcopenia.

Example 4. Construction of Muscle Atrophy Mouse Models and Analysis of Markers

[0117] To induce muscle atrophy in C57BL/6J male mice (the Laboratory Animal Resource Center at the Korea Research Institute of Bioscience and Biotechnology), the TA (tibialis anterior) muscle immobilization method was used (Caron A Z, J Appl Physiol. 106(6) 2049-2059, 2009). The principle used in this method is that when a leg is placed in a cast and the muscle of the leg is immobilized (i.e., not frequently used), the muscle is lost. It is a method for inducing muscle regeneration, in which, after the muscle loss due to immobilizing the shin muscle, the muscle is regenerated by releasing the immobilized muscle so that the muscle may move again. Specifically, the thighs and shins of both legs of mice were fixed using a medical staple so that legs were immobilized, and after leaving the immobilized mice alone for 5 days, the fixed legs were released, thereby constructing muscle atrophy mouse models.

[0118] In order to measure the change in serum protein levels by the induction of muscle atrophy, serum was isolated from the mice before immobilization of the mouse legs, on 5 days after immobilization, and on 2 and 4 days after release of the mouse legs, and the levels of tetranectin and gelsolin in the serum were analyzed using MyBioSource Elisa kits (Gelsolin, Cat # MBS2886136; Tetranectin, Cat # MBS2885296). The results are shown in FIG. 9.

[0119] As shown in FIG. 9, when muscle atrophy was induced by immobilization of the legs, the serum tetranectin and gelsolin levels all significantly increased compared to those before immobilization and during the recovery period (2 days and 4 days after release of the legs). These results indicate that tetranectin and gelsolin according to the present invention may be used as markers to detect muscle atrophy.

[0120] The above-described results suggest that muscle weakness-related disease can be effectively diagnosed with high accuracy by measuring tetranectin, gelsolin, or blood biomarkers including them.