Biomarker for diagnosing osteosarcopenia and use thereof

Abstract

The present invention relates to: a biomarker composition for diagnosing osteosarcopenia, comprising CSF-1, SLAMF1, PD-L1, CD40, OPG, IL-7, FGF-23, or IL-13; a composition for diagnosing osteosarcopenia; a kit; and a method for providing information for diagnosing osteosarcopenia, wherein the biomarkers simultaneously exhibit significance with bone mineral density-related clinical variables and muscle-related clinical variables, and thus can be effectively used for osteosarcopenia diagnosis using a biological sample such as blood.

Description

Diagnostic biomarkers for musculoskeletal disorders and their uses

[0001] The present invention relates to a biomarker composition for diagnosing musculoskeletal disease comprising CSF-1, SLAMF1, PD-L1, CD40, OPG, IL-7, FGF-23, or IL-13; a composition for diagnosing musculoskeletal disease; a kit; and a method for providing information for diagnosing musculoskeletal disease.

[0002]

[0003] Aging brings about physiological, neuromuscular, immunological, and endocrine changes in the body, which affect the biological and functional characteristics of skeletal muscle and bone. As the aging of the global population accelerates, managing musculoskeletal diseases in the elderly, such as sarcopenia and osteoporosis, is becoming increasingly important, as these can lead to negative health outcomes such as falls and mortality. Numerous studies have explored the co-occurrence of sarcopenia and osteoporosis, as well as their common risk factors and pathophysiological pathways. As a result, a new concept called "osteosarcopenia" has been introduced, characterized by the simultaneous occurrence of muscle weakness and bone quality decline. The prevalence of osteosarcopenia in the elderly is estimated at approximately 18.5%, and it is associated with a significant increase in falls, fractures, hospitalizations, institutionalizations, and mortality. According to some studies, osteosarcopenia exhibits unique clinical characteristics distinct from sarcopenia and osteoporosis; specifically, when observed in community-dwelling older adults, it displays features such as a decline in physical function and an increased risk of death compared to patients with sarcopenia or osteoporosis. Therefore, in an aging society, it is crucial to understand the pathophysiology of osteosarcopenia and identify its risk factors.

[0004] Various factors from genetic, mechanical, and endocrine perspectives have been proposed to explain the pathogenesis of osteoporosis and sarcopenia. Immunological abnormalities and chronic inflammation have been emphasized as key elements in the complex etiology of sarcopenia and osteoporosis. Recent studies have reported that skeletal muscle plays a crucial role in regulating immune processes and inflammatory responses, and the regenerative capacity of skeletal muscle is associated with the interactions between skeletal muscle, immune cells, and inflammatory cytokines. Various inflammatory signaling pathways can influence the bone remodeling process, and abnormalities in these pathways can affect the functions of osteoblasts and osteoclasts, ultimately leading to increased bone resorption and decreased bone formation. Therefore, the interaction between muscle-bone metabolism, immune abnormalities, and inflammation can be presumed to be a major etiological factor of osteosarcopenia.

[0005] Proteomics is a widely used approach for the discovery of biomarkers in various human diseases. The Proximity Extension Assay (PEA) is an advanced proteomics technology that enables precise protein profile analysis capable of simultaneously detecting hundreds of target proteins in a single sample. This technology has been successfully applied to various conditions, including depression, dementia, and ischemic stroke.

[0006] Accordingly, the inventors of the present invention completed the invention by discovering a biomarker that enables the diagnosis of osteomuscular loss.

[0007]

[0008] The object of the present invention is to provide a biomarker composition for the diagnosis of musculoskeletal disease comprising the CSF-1 protein or a gene encoding the same.

[0009] Another objective of the present invention is to provide a composition for diagnosing musculoskeletal disease comprising a preparation capable of measuring the expression level of the CSF-1 protein or the gene encoding it.

[0010] Another objective of the present invention is to provide a diagnostic kit for musculoskeletal disease comprising the above-mentioned composition for diagnosing musculoskeletal disease.

[0011] Another objective of the present invention is to provide a method for providing information for the diagnosis of musculoskeletal disease, comprising the steps of: (a) measuring the expression level of a CSF-1 protein isolated from a biological sample or a gene encoding the same; and (b) comparing the expression level of the CSF-1 protein or the gene encoding the same with a reference value obtained from a control sample.

[0012]

[0013] To achieve the above objective, the present invention provides a biomarker composition for the diagnosis of musculoskeletal disease comprising a CSF-1 protein or a gene encoding the same.

[0014] In addition, the present invention provides a composition for diagnosing osteomuscle loss comprising a preparation capable of measuring the expression level of the CSF-1 protein or the gene encoding it.

[0015] In addition, the present invention provides a diagnostic kit for osteomuscle loss comprising the above-mentioned composition for diagnosing osteomuscle loss.

[0016] In addition, the present invention provides a method for providing information for the diagnosis of osteomuscular loss, comprising the steps of: (a) measuring the expression level of a CSF-1 protein isolated from a biological sample or a gene encoding the same; and (b) comparing the expression level of the CSF-1 protein or the gene encoding the same with a reference value obtained from a control sample.

[0017]

[0018] The biomarker for diagnosing osteosarcopenia according to the present invention simultaneously exhibits significance with clinical variables related to bone density and clinical variables related to muscle, and thus can be usefully employed in the diagnosis of osteosarcopenia using biological samples such as blood.

[0019]

[0020] Figures 1a to 1e show the results of comparing the expression levels of 20 inflammation-related proteins identified as DEPs (Differentially expressed proteins) among inflammation-related proteins.

[0021] Figure 2a shows the results of analyzing proteins that show correlations with muscle-related and bone density-related clinical variables, 2b and 2c show the results of PCA and heatmap analysis for DEPs, and 2d shows the results of investigating interactions between proteins.

[0022] Figures 3 and 4 show the ROC curve analysis results of each biomarker.

[0023] Figure 5 shows the results of ROC curve analysis by biomarker combination.

[0024]

[0025] The present invention will be described in detail below.

[0026] The terms used in this invention have been selected based on currently widely used general terms whenever possible, taking into account the functions of the invention; however, these terms may vary depending on the intent of those skilled in the art or the emergence of new technologies. Additionally, in specific cases, terms may be selected arbitrarily, and in such cases, their meanings will be described in detail in the description section of the relevant embodiments. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0027] In the present invention, when a component or step is described as "comprising," this means that, unless specifically stated otherwise, it does not exclude other components or steps but may include additional components or steps.

[0028]

[0029] The present invention provides a biomarker composition for diagnosing musculoskeletal disease comprising the CSF-1 (macrophage colony-stimulating factor 1) protein or a gene encoding the same.

[0030] The term "diagnosis" in the present invention means confirming the existence or characteristics of a pathological condition. In the present invention, the diagnosis may be interpreted as confirming whether there is progression or onset of musculoskeletal disease.

[0031] In the present invention, the composition may further comprise a protein selected from the group consisting of SLAMF1 (signaling lymphocytic activation molecule 1), PD-L1 (Programmed cell death 1 ligand 1), CD40 (Tumor necrosis factor receptor superfamily member 5), and OPG (Tumor necrosis factor receptor superfamily member 11B), or a gene encoding therefrom. Additionally, the composition may further comprise a protein selected from the group consisting of IL-7 (Interleukin-7), FGF-23 (Fibroblast growth factor 23), and IL-13 (Interleukin-13), or a gene encoding therefrom.

[0032] In the present invention, it was confirmed that a protein selected from the group consisting of CSF-1, SLAMF1, PD-L1, CD40, OPG, IL-7, FGF-23, and IL-13, or a gene encoding such a protein, can be used as a biomarker for the diagnosis of osteosarcoma by simultaneously demonstrating significance with clinical variables related to bone density and clinical variables related to muscle.

[0033] In addition, the present invention provides a composition for diagnosing osteomuscle loss comprising a preparation capable of measuring the expression level of the CSF-1 protein or the gene encoding it.

[0034] In the present invention, the composition may further comprise a preparation capable of measuring the expression level of a protein selected from the group consisting of SLAMF1, PD-L1, CD40, and OPG, or a gene encoding therefrom. Additionally, the composition may further comprise a preparation capable of measuring the expression level of a protein selected from the group consisting of IL-7, FGF-23, and IL-13, or a gene encoding therefrom.

[0035] In the present invention, the preparation capable of measuring the expression amount of the protein may be selected from the group consisting of antibodies, oligopeptides, ligands, aptamers, and PNAs (peptide nucleic acids) that specifically bind to the protein or a fragment thereof, but is not limited thereto.

[0036] The above term, "antibody," means a specific immunoglobulin directed toward an antigenic site and includes polyclonal antibodies, monoclonal antibodies, recombinant antibodies, and combinations thereof. In addition, it includes polyclonal antibodies, monoclonal antibodies, recombinant antibodies, and complete forms having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules, e.g., Fab, F(ab'), F(ab')2, and Fv. Antibodies can be easily produced using techniques widely known in the field to which the present invention belongs, and commercially available antibodies can be used.

[0037] The term "aptamer" above refers to a type of polynucleotide composed of a special class of single-stranded nucleic acids (DNA, RNA, or modified nucleic acids) that possess a stable tertiary structure and can bind to target molecules with high affinity and specificity. Aptamers can be used as a substitute for antibodies because they are composed of polynucleotides that can specifically bind to antigenic substances in the same way as antibodies, yet are more stable than proteins, have a simple structure, and are easy to synthesize. Aptamers can be easily produced using techniques widely known in the field to which this invention belongs, and commercially available antibodies can be used.

[0038] The term "PNA" refers to an artificially synthesized polymer similar to DNA or RNA. While DNA has a phosphate-ribose sugar backbone, PNA has a repeating N-(2-aminoethyl)-glycine backbone connected by peptide bonds. This significantly increases the binding affinity and stability to DNA or RNA, and it is used in molecular biology, diagnostic analysis, and antisense therapy. The above PNA can be further specified by referring to the literature [Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991) "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254(5037): 1497-1500].

[0039] In the present invention, the agent capable of measuring the expression level of the gene may be selected from the group consisting of a primer, a probe, and an antisense oligonucleotide that specifically binds to the mRNA of the gene, but is not limited thereto.

[0040] The above term, "primer," refers to a short base sequence having a short free 3' hydroxyl group, capable of forming base pairs with a complementary template, and acting as a starting point for template strand replication. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerate or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature. The PCR conditions and the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the field to which the present invention belongs.

[0041] The term "probe" above refers to a nucleic acid fragment, such as RNA or DNA, capable of specifically binding to a gene, and is labeled to confirm the presence or absence and expression level of a specific gene. The probe may be produced in the form of an oligonucleotide probe, a single-strand DNA probe, a double-strand DNA probe, an RNA probe, etc. The selection of an appropriate probe and hybridization conditions may be appropriately selected according to techniques known in the field to which the present invention belongs.

[0042] The above term, "antisense oligonucleotide," refers to DNA, RNA, or derivatives thereof containing a nucleic acid sequence complementary to a specific mRNA sequence, which acts to inhibit the translation of the mRNA into a protein by binding to the complementary sequence within the mRNA. An antisense oligonucleotide sequence refers to a DNA or RNA sequence that is complementary to the mRNA of the said genes and capable of binding to said mRNA.

[0043] In addition, the present invention provides a diagnostic kit for osteomuscle loss comprising the above-mentioned composition for diagnosing osteomuscle loss.

[0044] In the present invention, the kit may be selected from the group consisting of RT-PCR kits, DNA chip kits, ELISA kits, protein chip kits, and rapid kits.

[0045] In addition to each primer set specific to the marker gene, the RT-PCR kit may include a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, etc. Additionally, it may include a primer set specific to the gene used as a quantitative control.

[0046] The above DNA chip kit may include a substrate to which cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, preparations, enzymes, etc. for producing a fluorescently labeled probe. Additionally, the substrate may include cDNA corresponding to a quantitative control gene or a fragment thereof.

[0047] The above ELISA kit may include a monoclonal antibody, a polyclonal antibody, or a recombinant antibody specific to a protein, and may also include reagents capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e.g., conjugated with an antibody) and its substrate, or other substances capable of binding to the antibody.

[0048] The above protein chip kit may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, a chromogenic substrate, etc. for the immunological detection of antibodies. In the above, the substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass, etc., the chromogenic enzyme may be peroxidase or alkaline phosphatase, the fluorescent substance may be FITC, RITC, etc., and the chromogenic substrate solution may be ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), or TMB (tetramethylbenzidine).

[0049] The above rapid kit is a kit capable of rapidly performing a test using a small amount of sample, and may use a substance capable of binding to an analyte contained in the sample, namely an antibody capable of specifically binding to a protein.

[0050] In addition, the present invention provides a method for providing information for the diagnosis of osteomuscular loss, comprising the steps of: (a) measuring the expression level of a CSF-1 protein isolated from a biological sample or a gene encoding the same; and (b) comparing the expression level of the CSF-1 protein or the gene encoding the same with a reference value obtained from a control sample.

[0051] In the present invention, the method may further include a step of determining that there is musculoskeletal loss when the expression level of the CSF-1 protein or the gene encoding it is higher than the reference value obtained from a control sample.

[0052] In the present invention, the method may further include the step of measuring the expression level of a protein selected from the group consisting of SLAMF1, PD-L1, CD40, and OPG, or a gene encoding therefrom. Additionally, the method may further include the step of determining that there is osteomyelopenia if the expression level of a protein selected from the group consisting of SLAMF1, PD-L1, CD40, and OPG, or a gene encoding therefrom, is higher than a reference value obtained from a control sample.

[0053] In the present invention, the method may further include the step of measuring the expression level of a protein selected from the group consisting of IL-7, FGF-23, and IL-13, or a gene encoding the same. Additionally, the method may further include the step of determining that there is osteomyelopenia if the expression level of a protein selected from the group consisting of FGF-23 and IL-13, or a gene encoding the same, is higher than a reference value obtained from a control sample, and the method may further include the step of determining that there is osteomyelopenia if the expression level of the IL-7 protein or a gene encoding the same is lower than a reference value obtained from a control sample.

[0054] In the present invention, the concentration of CSF-1 protein may be determined to be normal when it is less than the reference value of 297.6234 pg / mL and determined to be osteomyelopenia when it is greater than 396.9954 pg / mL, the concentration of SLAMF1 protein may be determined to be normal when it is less than the reference value of 47.8 pg / mL and determined to be osteomyelopenia when it is greater than 57.032089065 pg / mL, and the concentration of PD-L1 protein may be determined to be normal when it is less than the reference value of 26.52935 pg / mL and determined to be osteomyelopenia when it is greater than 28.9 pg / mL. In addition, the concentration of CD40 protein may be judged as normal when it is less than 899.558 pg / mL and as osteosarcopenia when it is greater than 969.8 pg / mL, and the concentration of OPG protein may be judged as normal when it is less than 556 pg / mL and as osteosarcopenia when it is greater than 816.2065 pg / mL. In addition, the concentration of IL-7 protein may be judged as normal when it is greater than 5.157557 pg / mL and as osteosarcopenia when it is less than 4.78 pg / mL.

[0055] In the present invention, the biological sample may be selected from the group consisting of tissue, cell, blood, serum, plasma, lymph fluid, saliva, and urine.

[0056] The mRNA expression level can be measured using reverse transcriptase polymerase chain reaction (RT-PCR), competitive reverse transcriptase polymerase chain reaction (competitive RT-PCR), real-time quantitative reverse transcriptase polymerase chain reaction (real-time quantitative RT-PCR), RNase protection method, Northern blotting, or DNA chip technology. In addition, the expression level of the above protein can be measured using western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, fluorescence-activated cell sorter analysis (FACS), or protein chip technology.

[0057]

[0058] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0059]

[0060] Example 1. Experimental Method

[0061] 1.1. Research Participants

[0062] Fifty patients with distal radial fractures requiring surgery (5 males, 45 females, mean age 70.8 ± 8.3 years, age range 55–90 years) were recruited from a tertiary hospital. For analysis, participants were divided into three groups based on the degree of osteoporosis. The control group consisted of subjects without sarcopenia or osteoporosis (n = 20), the sarcopenia group consisted of subjects with functional sarcopenia but without osteoporosis (n = 15), and the osteosarcopenia group consisted of subjects with both functional sarcopenia and osteoporosis (n = 15).

[0063] Participants with a history of conditions that may affect bone and muscle metabolism and activity, such as diabetes, liver disease, cancer, renal failure, rheumatoid arthritis, thyroid disease, or neurodegenerative disease, as well as those who have taken bone-activating drugs such as hormone regulators, anabolic agents, bisphosphonates, or denosumab, were excluded. Each participant's height, weight, calf and upper arm circumference were measured by a trained nurse. This study was approved by the Institutional Review Board of a tertiary hospital (Approval No.: KBSMC-2024-02-009) and was conducted after obtaining written informed consent from all participants.

[0064]

[0065] 1.2. Evaluation of Osteopenia

[0066] Sarcopenia was defined as functional sarcopenia, diagnosed when a grip strength of 18 kg or less was observed in women and 28 kg or less in men, along with a walking speed of less than 1.0 m / sec. Trained nurses measured hand grip strength using the uninjured contralateral hand, following the protocol described in previous studies. Each participant underwent three hand grip strength measurements, and the average value was calculated. In cases where the dominant hand was injured, grip strength was estimated using the 10% rule. Walking speed over a distance of 6 m was measured three times and recorded as the average value.

[0067] Bone mineral density (aBMD, areal bone mineral density, g / cm²) of the hip joint, femoral neck, and lumbar spine 2 ) was evaluated using a Hologic device (Horizon-W; Hologic Inc., Bedford, MA, USA). T-scores were evaluated according to the standard technique of the Korean Osteoporosis Society, and osteoporosis was diagnosed when the aBMD T-score of the femoral neck was -2.5 or lower. Osteosarcopenia was diagnosed when both functional sarcopenia and osteoporosis were present.

[0068]

[0069] 1.3. Statistical Analysis

[0070] The normality of the data was evaluated using the Shapiro-Wilk normality test, and parametric testing methods were applied after confirming that the data in this study followed a normal distribution. One-way ANOVA with Bonferroni correction was used for comparisons between continuous variables, while the chi-square test was used for categorical variables. The correlation between relative protein levels determined by PEA proteomic analysis and clinical variables related to muscle function or bone density was analyzed using Pearson correlation analysis, and the diagnostic accuracy of identifying sarcoma was evaluated through ROC curve analysis and AUC. For IL-7, which exhibits a negative fold change due to decreased expression in patients with sarcoma, ROC curve analysis was applied to evaluate its potential as a negative diagnostic indicator.

[0071]

[0072] Example 2. Demographic characteristics, bone density, and biochemical analysis results of study participants

[0073] First, the demographic characteristics of the participants in this study were analyzed. As a result, no significant differences were observed between groups in terms of age (P= 0.075), gender distribution (P= 0.499), BMI (P= 0.890), and upper arm circumference (P= 0.369) (Table 1). On the other hand, significant differences were observed in grip strength (P= 0.036), walking speed (P< 0.001), calf circumference (P= 0.037), and aBMD of the hip joint, femoral neck, and lumbar spine (P< 0.001) (Table 1).

[0074]

[0075] Total(n = 50)Control(n = 20)Sarcopenia(n = 15)Osteosarcopenia(n = 15)PvaluePost- hocAge (years)71.4 ± 7.470.2 ± 6.569.4 ± 9.975.0 ± 7.30.075Sex (male / female)†6 / 442 / 181 / 143 / 120.499BMI (body mass index)24.6 ± 2.724.7 ± 3.024.7 ± 2.724.3 ± 2.40.890Handgrip strength (kg)19.3 ± 7.421.3 ± 8.620.6 ± 6.215.2 ± 5.30.036*Control-OsteosarcopeniaGait speed (m / sec)0.87 ± 0.351.14 ± 0.260.80 ± 0.180.57 ± 0.29< 0.001**Control-Sarcopenia,Control-OsteosarcopeniaSarcopenia-OsteosarcopeniaUpper arm circumference26.8 ± 2.727.5 ± 2.726.7 ± 2.426.1 ± 3.10.369Calf circumference32.8 ± 2.232.7 ± 1.633.8 ± 2.331.7 ± 2.50.037*Sarcopenia-OsteosarcopeniaUnderlying diseases (no / yes)†Hypertension33 / 1714 / 611 / 48 / 70.455Diabetes Mellitus42 / 817 / 312 / 313 / 20.872Heart diseases43 / 716 / 415 / 012 / 30.175Liver diseases49 / 120 / 014 / 115 / 00.304Respiratory diseases48 / 219 / 114 / 115 / 00.620Neurologic disorders44 / 619 / 113 / 212 / 30.394Dyslipidemia47 / 318 / 215 / 014 / 10.464Total hip aBMD (g / cm 2)0.718 ± 0.1310.800 ± 0.0820.745 ± 0.0670.583 ± 0.126< 0.001**Control-Osteosarcopenia, Sarcopenia-OsteosarcopeniaTotal hip T score-1.252 ± 1.19-0.510 ± 0.693-1.000 ± 0.614-2.493 ± 1.188< 0.001**Control-Osteosarcopenia, Sarcopenia-OsteosarcopeniaFemoral neck aBMD (g / cm 2 )0.670 ± 0.0730.670 ± 0.0730.621 ± 0.0670.491 ± 0.077< 0.001**Control-Osteosarcopenia, Sarcopenia-OsteosarcopeniaFemoral neck T score-1.92 ± 0.97-1.27 ± 0.68-1.77 ± 0.62-2.93 ± 0.73< 0.001**Control-Osteosarcopenia, Sarcopenia-Osteosarcopenialumbar spine 1-4 aBMD (g / cm 2)0.823 ± 0.1460.874 ± 0.1280.846 ± 0.1610.735 ± 0.1180.003*Control-Osteosarcopenia, Sarcopenia-Osteosarcopenialumbar spine 1-4 T score-1.648 ± 1.186-1.170 ± 1.116-1.473 ± 1.039-2.460 ± 1.0400.014*Control-Osteosarcopenia, Sarcopenia-OsteosarcopeniaWBC ( / uL)7.89 ± 1.847.37 ± 1.388.68 ± 1.857.79 ± 2.180.108RBC ( / uL)4.13 ± 0.424.12 ± 0.474.24 ± 0.364.24 ± 0.360.447Hemoglobin (g / dL)12.7 ± 1.312.5 ± 1.312.8 ± 1.313.0 ± 1.50.542Total protein (g / dL)7.09 ± 0.506.90 ± 0.357.25 ± 0.517.17 ± 0.610.092Albumin (g / dL)4.49 ± 0.304.46 ± 0.234.61 ± 0.334.43 ± 0.340.209ESR (mm / hr)22.8 ± 18.818.9 ± 15.924.1 ± 22.527.1 ± 18.70.438CRP (mg / dL)0.454 ± 0.6150.659 ± 0.8680.286 ± 0.3040.350 ± 0.3240.153CTX (ng / mL)0.443 ± 0.2730.361 ± 0.2080.481 ± 0.2840.515 ± 0.3560.214Osteocalcin (ng / mL)18.4 ± 8.116.4 ± 7.019.9 ± 6.619.6 ± 10.50.367IGF-1 (ng / mL)105.1 ± 36.5101.0 ± 35.7118.6 ± 45.797.1 ± 23.30.221PTH (pg / mL)44.6 ± 20.740.3 ± 17.240.8 ± 15.354.2 ± 26.90.097Cortisol (mcg / dL)11.6 ± 6.211.9 ± 5.013.3 ± 8.79.62 ± 4.260.27125 (OH)-hydroxyvitamin D3 (ng / mL)32.7 ± 18.540.8 ± 21.625.6 ± 15.429.0 ± 12.70.032*Control-Sarcopenia.

[0076] - Values ​​are expressed as mean ± SD; data were obtained by one-way ANOVA with Bonferroni correction; † data were obtained by chi-square experiment; and* P< 0.05, **P< 0.001.

[0077]

[0078] Next, biochemical parameters were analyzed from the peripheral venous blood of the study participants. Peripheral venous blood samples from each participant were collected between 7:00 AM and 11:00 AM on an empty stomach to minimize circadian variability. The concentrations of white blood cell count, red blood cell count, hemoglobin, total protein, albumin, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), C-telopeptide of type I collagen (CTX), osteocalcin, insulin-like growth factor-1 (IGF-1), parathyroid hormone (PTH), cortisol, and vitamin D3 (25(OH)-hydroxyvitamin D3) were analyzed in the collected blood.

[0079] As a result, no significant differences were observed in WBC (P= 0.108), RBC (P= 0.447), hemoglobin (P= 0.542), total protein (P= 0.092), albumin (P= 0.209), ESR (P= 0.438), CRP (P= 0.153), CTX (P= 0.214), osteocalcin (P= 0.367), IGF-1 (P= 0.221), PTH (P= 0.097), and cortisol (P= 0.271), whereas vitamin D3 levels showed a significant difference between the control group and the sarcopenia group (P= 0.032) (Table 1).

[0080]

[0081] Example 3. Proteomic analysis using PEA (Proximity extension assay)

[0082] 3.1. Method

[0083] Proteomic analysis using the Proximity Extension Assay (PEA) was performed using 20 μL of plasma collected between 7:00 AM and 11:00 AM. Inflammation-related protein levels were quantified using the Olink PEA Inflammation Panel (Macrogen, Seoul), and 92 specific proteins were targeted. Each target protein was identified via dual antibody labeling, subsequently bound to an auxiliary DNA barcode, and quantified via high-speed microfluidic real-time polymerase chain reaction (PCR). The results were expressed as Normalized Protein Expression Values ​​(NPX), a relative quantification unit with a logarithmic relationship to protein concentration, and these values ​​were converted to a log2 scale.

[0084]

[0085] 3.2. Results of Inflammation-Related Protein Analysis

[0086] As a result of measuring the expression levels of 92 inflammation-related proteins in three groups, a total of 20 inflammation-related proteins were identified as DEPs (Differentially expressed proteins). Among them, 19 proteins, including OPG, uPA, MCP-1, CXCL11, CXCL9, OSM, SLAMF1, TNFSF14, FGF-23, PD-L1, IL13, MMP-10, CD5, CXCL10, IL4, TWEAK, TNFB, and CSF-1, showed increased expression, while IL-7 showed decreased expression in patients with osteosarcopenia (Figs. 1a to 1e). Post-hoc analysis revealed that among these, OPG, uPA, MCP-1, CXCL11, CXCL9, SLAMF1, FGF-23, PD-L1, IL-13, MMP-10, CD5, CXCL10, and TWEAK showed significant differences between the control and osteosarcopenia groups, while IL-7, OSM, CD40, and IL-4 showed significant differences between the sarcopenia and osteosarcopenia groups. MCP-1, CXCL11, PD-L1, CXCL10, and CSF-1 showed significant differences in both the control and osteosarcopenia groups, as well as between the sarcopenia and osteosarcopenia groups.

[0087]

[0088] 3.3. Analysis Results of Proteins Related to Muscle Function and Bone Density

[0089] A total of 33 proteins were identified that showed significant correlations with clinical variables. TSLP, CXCL5, IL-7, IL-4, IL-2, and IL-33 showed significant correlations only with muscle function-related variables such as grip strength, calf circumference, and walking speed, while proteins such as CXCL11, CXCL9, LIF-R, CXCL10, MMP-10, CD5, MCP-1, TGF-α, uPA, and FGF-5 showed significant associations with bone density variables. SLAMF1, OPG, FGF-23, IL-10RB, CCL20, PD-L1, CCL25, TNFRSF9, CX3CL1, GDNF, CSF-1, CDCP1, MCP-3, IL-13, CD40, CXCL6, and IL-15RA showed significant correlations with both bone density and muscle function-related variables (Fig. 2a). A total of 37 proteins were identified as either DEPs or showing a significant correlation with muscle function-related variables. Among them, SLAMF1, OPG, FGF-23, PD-L1, CSF-1, IL-13, IL-7, and CD40 belong to the DEPs and were confirmed to show simultaneous significance with muscle-related clinical variables and bone density-related clinical variables in Pearson correlation analysis.

[0090] From the above results, it was confirmed in the present invention that SLAMF1, OPG, FGF-23, PD-L1, CSF-1, IL-13, IL-7, or CD40 can be used as biomarkers for the diagnosis of osteomyelopenia.

[0091]

[0092] Example 4. Additional Analysis of DEPs and Protein Network

[0093] 4.1. Method

[0094] Data normalization and identification of Differentially Expressed Proteins (DEPs) across groups were performed using the "OlinkAnalyze" package in R (R Core Team, 2020, Version 4.2.2). Principal Component Analysis (PCA) using the 'factoextra' package was applied to classify participants according to their protein expression profiles, and a heatmap containing DEPs was generated using the 'Complexheatmap' package. Further analysis of DEPs was conducted to investigate local network clusters and signaling pathways contributing to the development of sarcopenia. Protein interactions and systematic functions were analyzed using the STRING database version 12.0.

[0095]

[0096] 4.2. Results of Additional DEP Analysis

[0097] Further analysis of DEPs was performed to investigate the unique characteristics of osteosarcopenia. PCA and heatmap analysis confirmed that the osteosarcopenia group formed an independent group compared to sarcopenia and the control group, whereas the sarcopenia group did not show a significant difference from the control group (Figs. 2b and 2c).

[0098]

[0099] 4.3. Protein Network Analysis Results

[0100] As a result of investigating protein-protein interactions using the STRING database version 12.0, a total of 12 major pathways were identified (Table 2). The STRING protein-protein interaction analysis revealed interactions including chemokine binding, IL-2, IL-4, IL-13, IL-7-related signaling, and TNF receptor activation, and confirmed that MCP-1, CD40, and PD-L1 were the most active protein-protein interaction nodes (Fig. 2d and Table 2).

[0101] As shown in Table 2, in an additional network analysis of eight proteins that are DEPs and showed significant correlations with muscle function and bone density, two major networks were identified: the 'T cell modulation in pancreatic cancer, and Lymphoproliferative syndrome 2' and the 'Mixed, incl. Adaptive immunity, and TNFs bind their physiological receptors' pathways, confirming that they may have a significant impact on the development of osteosarcopenia.

[0102]

[0103] Cluster descriptionFDRMoleculesChemokine receptors bind chemokines< 0.001**MCP-1, CXCL10, CXCL11, CXCL9CXC Chemokine domain, and Regulation of dendritic cell dendrite assembly< 0.001**MCP-1, CXCL10, CXCL9CXCR3 chemokine receptor binding, and CCR4 chemokine receptor binding0.0043MCP-1, CXCL9Interleukin-2 family signaling, and Interleukins 4 and 130.0017IL-13, IL-7, IL-4interleukin-7-mediated signaling pathway, and Interleukins 4 and 130.0043IL-7, IL-4JAK-STAT signaling pathway< 0.001**IL-13, IL-7, IL-4, OSMMixed, incl. Adaptive immunity, and TNFs bind their physiological receptors< 0.001**CD5, CD274(PD-L1), CD40, SLAMF1, TNFSF14Mixed, incl. Chemokine-mediated signaling pathway, and Adaptive immunity< 0.001**MCP-1, CD5, CXCL9, CXCL10, CXCL11, PD-L1, CD40, SLAMF1, TNFSF14Mixed, incl. T cell modulation in pancreatic cancer, and non-Langerhans-cell histiocytosis< 0.001**PD-L1, CD40, TNFSF14T cell modulation in pancreatic cancer, and Lymphoproliferative syndrome 20.0299PD-L1, CD40TNF receptor superfamily (TNFSF) members mediating non-canonical NF-kB pathway, and Glial cell-neuron signaling0.0067LTA, TWEAKTNFs bind their physiological receptors, and T cell modulation in pancreatic cancer0.0010PD-L1, CD40, TNFSF14.

[0104] - FDR, false discovery rate; and significance: FDR < 0.05 (*FDR < 0.05, ** FDR < 0.001).

[0105]

[0106] Example 5. ELISA Cross-validation

[0107] 5.1. Method

[0108] Serum samples collected from 50 participants were cross-validated using ELISA. Proteins that are DEPs and show significant correlations with variables associated with muscle function and aBMD of the hip, femoral neck, or lumbar spine were selected, and cross-validation was performed using the ELISA method. Serum levels of eight proteins (CD40, IL-13, IL-7, PD-L1, FGF-23, OPG, SLAMF1, and CSF-1) identified as potential biomarkers for osteosarcopenia were measured and quantified using an ELISA kit, and all experiments were repeated three times to derive the average data.

[0109]

[0110] 5.2. Results of Protein Expression Verification by ELISA

[0111] As a result of measuring the concentrations of eight proteins in plasma using an ELISA kit, CD40, IL-7, PD-L1, OPG, SLAMF1, and CSF-1 proteins showed significant differences in serum concentrations among the three groups, whereas no significant differences were observed in the serum concentrations of IL-13 and FGF-23 (Table 3). In particular, as shown in Table 4, Figure 3, and Figure 4, CSF-1 was confirmed to exhibit the highest diagnostic accuracy at the optimal cutoff value, with an AUC of 0.874, a sensitivity of 84.2%, and a specificity of 82.6%. Additionally, PD-L1 showed an AUC of 0.867, a sensitivity of 75%, and a specificity of 73.3%, while CD40 showed an AUC of 0.847, a sensitivity of 75%, and a specificity of 73.3%. IL-7 showed an AUC of 0.725, sensitivity of 69.2%, and specificity of 71.4% as a negative biomarker, and SLAMF1 showed significant diagnostic power with an AUC of 0.764, sensitivity of 75%, and specificity of 73.1%. However, IL-13 (AUC 0.442, sensitivity 38.5%, specificity 62.1%) and FGF-23 (AUC 0.595, sensitivity 58.8%, specificity 48%) did not show significant results.

[0112]

[0113] Total(n = 50)Control(n = 20)Sarcopenia(n = 15)Osteosarcopenia(n = 15)PvaluePost- hocCD40 (pg / mL)941.4 ± 321.4805.6 ± 269.6884.4 ± 177.31221.8 ± 326.2< 0.001**Control-OsteosarcopeniaIL-13 (pg / mL)273.8 ± 31.3266.1 ± 3.7269.7 ± 19.2288.4 ± 52.30.120IL-7 (pg / mL)5.90 ± 2.826.63 ± 3.116.86 ± 3.114.23 ±1.050.028*Control-OsteosarcopeniaPD-L1 (pg / mL)29.6 ± 12.522.7 ± 4.629.6 ± 10.941.9 ± 14.2< 0.001**Control-Sarcopenia,Control-OsteosarcopeniaFGF-23 (pg / mL)86.5 ± 167.454.0 ± 37.076.9 ± 68.1123.6 ± 256.50.483OPG (pg / mL)904.8 ± 359.5767.6 ± 285.7756.8 ± 286.01158.9 ± 353.5< 0.001**Control-Osteosarcopenia, Sarcopenia-OsteosarcopeniaSLAMF1 (pg / mL)105.3 ± 141.158.1 ± 67.370.9 ± 61.3177.1 ± 196.20.030*Control-Osteosarcopenia,CSF-1 (pg / mL)475.1 ± 411.8241.3 ± 133.6272.8 ± 117.4744.9 ± 475.9< 0.001**Sarcopenia-Osteosarcopenia,Control-Osteosarcopenia

[0114] - Values ​​are expressed as mean ± SD; data were obtained by one-way analysis of variance (ANOVA) with Bonferroni correction; and* P< 0.05, **P< 0.001.

[0115]

[0116] OptimalCutoff value(pg / mL)†AUC†Pvalue†Clinimetric valuesSensitivity %Specificity %CD40969.80.847< 0.001**75.073.3IL-13264.20.4420.54938.562.1IL-7‡4.780.7250.020*69.271.4PD-L128.90.867< 0.001**75.073.3FGF-2342.90.5950.29958.848.0OPG556.00.804< 0.001**86.781.5SLAMF153.20.7640.004*75.073.1CSF-1331.30.874< 0.001**84.282.6

[0117] - Values ​​listed are expressed as mean ± SD; †Data were obtained via ROC curve analysis; ‡ROC curve analysis was applied to IL-7 to evaluate its potential as a negative diagnostic indicator; and* P< 0.05, **P< 0.001.

[0118]

[0119] Example 6. Diagnosis of osteosarcoma by biomarkers

[0120] In the present invention, the diagnostic range of osteosarcopenia based on eight biomarkers was determined according to the results of Tables 3 and 4 above. As a result, as shown in Table 5 below, it was confirmed that CSF-1 concentrations below 297.6234 pg / mL are judged as normal, and osteosarcopenia above 396.9954 pg / mL is judged as osteosarcopenia. In addition, it was confirmed that SLAMF1 concentrations below 47.8 pg / mL are judged as normal, and osteosarcopenia above 57.032089065 pg / mL is judged as osteosarcopenia, and PD-L1 concentrations below 26.52935 pg / mL are judged as normal, and osteosarcopenia above 28.9 pg / mL is judged as osteosarcopenia. In addition, it was confirmed that a CD40 concentration of less than 899.558 pg / mL is considered normal and greater than 969.8 pg / mL is considered sarcopenia, and that an OPG concentration of less than 556 pg / mL is considered normal and greater than 816.2065 pg / mL is considered sarcopenia. In addition, it was confirmed that an IL-7 concentration of greater than 5.157557 pg / mL is considered normal and less than 4.78 pg / mL is considered sarcopenia.

[0121]

[0122] NormalSarcopeniaOsteosarcopeniaCD40 (pg / mL)< 899.558899.558-969.8969.8 <IL-7 (pg / mL)5.157557 <4.78-5.1575574.78 >PD-L1 (pg / mL)< 26.5293526.52935-28.928.9 <OPG (pg / mL)< 556556-816.2065816.2065 <SLAMF1 (pg / mL)< 47.847.8-57.0320890757.032089065<CSF-1 (pg / mL)< 297.6234297.6234-396.9954396.9954 <

[0123] - Values ​​listed are expressed as mean ± SD; data were obtained using one-way ANOVA with Bonferroni correction; † data were obtained using ROC curve analysis; ‡ ROC curve analysis was applied to IL-7 to evaluate its potential as a voice diagnostic indicator; and* P< 0.05, **P< 0.001.

[0124]

[0125] Example 7. Verification of sensitivity and specificity of osteosarcopenia by biomarker combination

[0126] Experiments were conducted to determine whether the sensitivity and specificity of osteosarcopenia could be improved through the combination of biomarkers of the present invention. As a result, as shown in Table 6 and Figure 5 below, the CSF-1 biomarker has a sensitivity of 84.2% and a specificity of 82.6%, making it possible to diagnose osteosarcopenia even when used alone. However, when OPG, CD40, and PD-L1 biomarkers or SLAMF1, OPG, CD40, and PD-L1 biomarkers were added in addition to CSF-1, the AUC increased to 0.950, the sensitivity to 96.7%, and the specificity to 83.3%, confirming that it can be used more effectively for the diagnosis of osteosarcopenia.

[0127]

[0128] No.MoleculesAUCSensitivity (%)Specificity (%)1CSF-10.87484.282.62CSF-1, OPG, CD40, PD-L10.950096.783.33CSF-1, SLAMF1, OPG, CD40, PD-L10.950096.783.3

[0129]

[0130] From the above results, it was confirmed that the present invention can be usefully employed for the diagnosis of musculoskeletal disease using a biomarker selected from the group consisting of CSF-1, SLAMF1, PD-L1, CD40, OPG, IL-7, FGF-23, and IL-13.

[0131]

[0132] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0133] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

1. A biomarker composition for the diagnosis of osteomyelopenia comprising CSF-1 (macrophage colony-stimulating factor 1) protein or a gene encoding the same.

2. In Paragraph 1, The above composition is a biomarker composition further comprising a protein selected from the group consisting of SLAMF1 (signaling lymphocytic activation molecule 1), PD-L1 (Programmed cell death 1 ligand 1), CD40 (Tumor necrosis factor receptor superfamily member 5), and OPG (Tumor necrosis factor receptor superfamily member 11B) or a gene encoding the same.

3. In Paragraph 1 or 2, The above composition is a biomarker composition further comprising a protein selected from the group consisting of IL-7 (Interleukin-7), FGF-23 (Fibroblast growth factor 23), and IL-13 (Interleukin-13), or a gene encoding the same.

4. A composition for diagnosing osteomyelopenia comprising a preparation capable of measuring the expression level of CSF-1 protein or the gene encoding it.

5. In Paragraph 4, A diagnostic composition comprising, wherein the above composition further comprises a preparation capable of measuring the expression level of a protein selected from the group consisting of SLAMF1, PD-L1, CD40, and OPG, or a gene encoding such protein.

6. In Paragraph 4 or 5, A diagnostic composition comprising, further comprising a preparation capable of measuring the expression level of a protein selected from the group consisting of IL-7, FGF-23, and IL-13, or a gene encoding the same.

7. In Paragraph 4, A diagnostic composition capable of measuring the expression amount of the above protein, wherein the preparation is selected from the group consisting of antibodies, oligopeptides, ligands, aptamers, and PNAs (peptide nucleic acids) that specifically bind to the above protein or a fragment thereof.

8. In Paragraph 4, A diagnostic composition capable of measuring the expression level of the above gene, wherein the preparation is selected from the group consisting of a primer, a probe, and an antisense oligonucleotide that specifically binds to the mRNA of the above gene.

9. A diagnostic kit for osteomuscle loss comprising a diagnostic composition according to any one of claims 4 to 8.

10. In Paragraph 9, The above kit is a kit selected from the group consisting of RT-PCR kits, DNA chip kits, ELISA kits, protein chip kits, and rapid kits. 11.(a) a step of measuring the expression level of the CSF-1 protein isolated from a biological sample or the gene encoding it; and (b) A method for providing information for the diagnosis of osteomuscular loss, comprising the step of comparing the expression level of the CSF-1 protein or the gene encoding it with a reference value obtained from a control sample.

12. In Paragraph 11, The above method is an information provision method that further includes the step of determining that there is osteomuscular loss when the expression level of the CSF-1 protein or the gene encoding it is higher than the reference value obtained from a control sample.

13. In Paragraph 11, The above method is an information-providing method that further includes the step of measuring the expression level of a protein selected from the group consisting of SLAMF1, PD-L1, CD40, and OPG, or a gene encoding the same.

14. In Paragraph 13, The above method is an information-providing method that further includes the step of determining that there is osteomuscular loss when the expression level of a protein selected from the group consisting of SLAMF1, PD-L1, CD40, and OPG, or a gene encoding the same, is higher than the reference value obtained from a control sample.

15. In Paragraph 11 or Paragraph 13, The above method is an information-providing method that further includes the step of measuring the expression level of a protein selected from the group consisting of IL-7, FGF-23, and IL-13, or a gene encoding the same.

16. In Paragraph 15, The above method is an information-providing method that further includes the step of determining that there is osteomuscular loss when the expression level of a protein selected from the group consisting of FGF-23 and IL-13 or a gene encoding the same is higher than the reference value obtained from a control sample.

17. In Paragraph 15, The above method is an information-providing method that further includes the step of determining that there is osteomyelopenia when the expression level of the IL-7 protein or the gene encoding it is lower than the reference value obtained from a control sample.

18. In Paragraph 11, A method for providing information, wherein the biological sample is selected from the group consisting of tissue, cell, blood, serum, plasma, lymph fluid, saliva, and urine.

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