Biomarkers for use in diagnosis of sarcopenia and the method of diagnosing sarcopenia using the same

Abstract

The present invention relates to compositions for the diagnosis of sarcopenia, and was completed by discovering biomarkers capable of diagnosing sarcopenia with high accuracy and sensitivity through qualitative and quantitative analysis of the proteome of an animal model of sarcopenia. In particular, it has been confirmed that the biomarkers according to the present invention have positive or negative correlations with sarcopenia-related indicators such as muscle mass, grip strength, Rotarod index, and inverted strength. Therefore, sarcopenia can be diagnosed through molecular diagnosis using the markers of the present invention, instead of conventional methods that relied on measuring muscle mass and muscle function; furthermore, it is possible to determine the severity of sarcopenia through the association between the markers and muscle function. In particular, biomarkers showing a consistent correlation with sarcopenia indicators are expected to be utilized as targets for the treatment of sarcopenia. Moreover, since sarcopenia is a disease closely related to aging, the biomarkers according to the present invention are expected to serve as a foundation for securing anti-aging treatment targets and developing aging control technologies.

Description

Technology Field

[0001] The present invention relates to a biomarker for diagnosing sarcopenia and a method for diagnosing sarcopenia using the same. Background Technology

[0002] Sarcopenia is a condition characterized by a decline in muscle strength and physical function due to a decrease in skeletal muscle mass. It is an official disease that received a diagnostic code in the United States (ICD-10-CM) in 2016 and in Korea (KCD-8) in 2021. While the causes of sarcopenia are diverse, the most common are reduced protein intake and a lack of exercise. In particular, a very high rate of sarcopenia is associated with insufficient intake and absorption of essential amino acids. Another common cause of sarcopenia is hormone deficiency associated with aging. Skeletal muscle is the largest organ in the human body, accounting for 40 to 50% of its mass. Skeletal muscle tends to decrease by approximately 1% annually starting from age 30, and declines rapidly after age 65. As aging progresses, the functional capacity of muscles gradually decreases; sarcopenia is diagnosed when both muscle strength and function decline along with a decrease in muscle mass.

[0003] Muscles are not only vital as motor organs but also affect the entire body, including bones, blood vessels, the heart, and nerves. In particular, since bones are moved and maintain their density through muscles, weakening muscles leads to weakened bones, making one susceptible to osteoporosis. Furthermore, a decrease in muscle mass results in a reduction or overproduction of various factors produced by muscles, hindering the formation of new blood vessels and nerves and ultimately potentially causing serious conditions such as cognitive decline. In addition, muscle loss is closely linked to diseases such as fatty liver, cardiac hypertrophy, pancreatic dysfunction, and diabetes.

[0004] It is crucial to detect and manage sarcopenia early before it progresses to a severe stage. By the time individuals reach their 70s, muscle mass decreases by approximately 30% compared to their 30s and 40s; however, since fat fills the void left by lost muscle, maintaining body weight, this muscle loss often goes unnoticed. When muscle mass declines excessively and is accompanied by a deterioration in joint function or overall physical condition, treatment through nutrition or exercise therapy may be difficult. Currently, sarcopenia is diagnosed by assessing muscle mass, strength, and function. Muscle mass is measured using methods such as dual-energy X-ray absorptiometry, bioimpedance analysis, CT, and MRI; however, these methods have limitations as diagnostic tools due to the low correlation between skeletal muscle mass and strength. Muscle strength is measured by leg strength or grip strength, while muscle function is assessed by performing one or two tests among walking speed, the Short Physical Performance Battery (SPPB), the 400-meter walk test, and the 6-minute walk test. However, since normal muscle mass values ​​vary depending on age, gender, height, body weight, and body fat percentage, the criteria for assessing muscle strength and function are not precise. Furthermore, performing such tests poses a problem as it is burdensome for the elderly.

[0005] Therefore, there is a need for a new diagnostic method that can easily determine the presence of sarcopenia based on more objective criteria. Prior art literature

[0006] Republic of Korea Published Patent Application No. 10-2022-0147428 The problem to be solved

[0007] The present invention was devised to solve the aforementioned problems and was completed by discovering a muscle tissue protein biomarker that exhibits a correlation with functional characteristics related to sarcopenia.

[0008] Accordingly, the object of the present invention is to provide a composition for diagnosing sarcopenia comprising, as an active ingredient, a preparation for measuring the protein or mRNA levels of the biomarkers.

[0009] Another objective of the present invention is to provide a kit for diagnosing sarcopenia comprising a preparation for measuring the protein or mRNA levels of the biomarkers.

[0010] Another objective of the present invention is to provide a method for diagnosing sarcopenia and a method for providing information therefor, comprising the step of measuring the protein or mRNA levels of the biomarkers.

[0011] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below. means of solving the problem

[0012] The present invention provides a composition for diagnosing sarcopenia comprising, as an active ingredient, a preparation for measuring the level of one or more proteins or mRNA selected from the group consisting of Asporin, PADI2 (Protein-arginine deiminase type-2), Adenosin 5'-monophosphoramidase HINT2, and HSDL2 (Hydroxysteroid dehydrogenase-like protein 2).

[0013] In one embodiment of the present invention, the protein or mRNA level of Asporin or PADI2 may be characterized as being increased in patients with sarcopenia compared to normal individuals, but is not limited thereto.

[0014] In another embodiment of the present invention, the protein or mRNA levels of the Adenosin 5'-monophosphoramidase HINT2 or HSDL2 may be characterized as being reduced in patients with sarcopenia compared to normal individuals, but are not limited thereto.

[0015] 본 발명의 또 다른 구현예에서, 상기 조성물은 Aldehyde dehydrogenase 1A1, Synaptogyrin-2, Troponin T, Pyruvate carboxylase, mitochondrial, Annexin A3, Heterogeneous nuclear ribonucleoproteins C1 / C2, Ribosome-binding protein 1, Ig gamma-3 chain C region, Ornithine aminotransferase (mitochondrial), Four and a half LIM domains protein 3, Threonine--tRNA ligase 1 (cytoplasmic), Striatin-3, Protein unc-45 homolog B, Heat shock 70 kDa protein 4, Long-chain specific acyl-CoA dehydrogenase (mitochondrial), Peptidyl-prolyl cis-trans isomerase D, Actin-binding LIM protein 1, AMP deaminase 3, Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (mitochondrial), ATP-binding cassette sub-family C member 9, Sorting nexin-3, Serine / threonine-protein phosphatase PGAM5 (mitochondrial), Eukaryotic peptide chain release factor GTP-binding subunit ERF3A, Dual specificity mitogen-activated protein kinase kinase 3, Long-chain fatty acid transport protein 1, Valacyclovir hydrolase, Myozenin-3,It may further include, but is not limited to, agents for measuring the level of one or more proteins or mRNA selected from the group consisting of [Pyruvate dehydrogenase (acetyl-transferring)] inase isozyme 4 (mitochondrial), and NADPH-cytochrome P450 reductase.

[0016] In another embodiment of the present invention, the agent for measuring the protein level may be an antibody or aptamer specific to the protein, but is not limited thereto.

[0017] In another embodiment of the present invention, the agent for measuring the mRNA expression level may be a set of primers or a probe that specifically binds to the mRNA, but is not limited thereto.

[0018] In another embodiment of the present invention, the protein or mRNA level may be, but is not limited to, a level in one or more selected from the group consisting of blood, whole blood, plasma, lymph fluid, urine, saliva, tissue, muscle tissue, cell, organ, bone marrow, fine needle aspiration specimen, core needle biopsy specimen, and vacuum aspiration biopsy specimen.

[0019] In addition, the present invention provides a kit for diagnosing sarcopenia comprising a composition according to the present invention.

[0020] In addition, the present invention provides a method for diagnosing sarcopenia and / or a method for providing information for diagnosing sarcopenia, comprising the following steps:

[0021] (S1) A step of measuring the level of one or more proteins or mRNA selected from the group consisting of Asporin, PADI2, Adenosin 5'-monophosphoramidase HINT2, and HSDL2 in a biological sample isolated from a subject; and

[0022] (S2) A step of comparing the protein or mRNA level of the subject measured in the above step (S1) with a control group.

[0023] In one embodiment of the present invention, the method may further include, but is not limited to, a step of determining that there is sarcopenia or a risk of developing sarcopenia if the protein or mRNA level of Asporin or PADI2 measured in step (S1) is higher than that of a control group.

[0024] In another embodiment of the present invention, the method may further include, but is not limited to, a step of determining that there is sarcopenia or that there is a risk of developing sarcopenia if the protein or mRNA level of Adenosin 5'-monophosphoramidase HINT2 or HSDL2 (hydroxysteroid dehydrogenase-like protein 2) measured in step (S1) is lower than that of a control group.

[0025] 본 발명의 또 다른 구현예에서, 상기 방법은 상기 피검체로부터 분리된 생물학적 시료에서 Aldehyde dehydrogenase 1A1, Synaptogyrin-2, Troponin T, Pyruvate carboxylase, mitochondrial, Annexin A3, Heterogeneous nuclear ribonucleoproteins C1 / C2, Ribosome-binding protein 1, Ig gamma-3 chain C region, Ornithine aminotransferase (mitochondrial), Four and a half LIM domains protein 3, Threonine--tRNA ligase 1 (cytoplasmic), Striatin-3, Protein unc-45 homolog B, Heat shock 70 kDa protein 4, Long-chain specific acyl-CoA dehydrogenase (mitochondrial), Peptidyl-prolyl cis-trans isomerase D, Actin-binding LIM protein 1, AMP deaminase 3, Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (mitochondrial), ATP-binding cassette sub-family C member 9, Sorting nexin-3, Serine / threonine-protein phosphatase PGAM5 (mitochondrial), Eukaryotic peptide chain release factor GTP-binding subunit ERF3A, Dual specificity mitogen-activated protein kinase kinase 3, Long-chain fatty acid transport protein 1, Valacyclovir hydrolase, Myozenin-3,The method may further include, but is not limited to, a step of measuring the level of one or more proteins or mRNA selected from the group consisting of [Pyruvate dehydrogenase (acetyl-transferring)] inase isozyme 4 (mitochondrial), and NADPH-cytochrome P450 reductase.

[0026] In another embodiment of the present invention, the biological sample may be one or more selected from the group consisting of blood, whole blood, plasma, lymph fluid, urine, saliva, tissue, muscle tissue, cell, organ, bone marrow, fine needle aspiration specimen, core needle biopsy specimen, and vacuum aspiration biopsy specimen, but is not limited thereto.

[0027] In another embodiment of the present invention, the protein level may be measured by one or more methods selected from the group consisting of protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, Ouchteroni immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry), Western blotting, ELISA (enzyme linked immunosorbent assay), and FACS, but is not limited thereto.

[0028] In another embodiment of the present invention, the mRNA level may be measured by one or more methods selected from the group consisting of PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization, DNA chip, and RNA chip, but is not limited thereto.

[0029] In addition, the present invention provides the use of a preparation for measuring the level of one or more proteins or mRNA selected from the group consisting of Asporin, PADI2, Adenosin 5'-monophosphoramidase HINT2, and HSDL2, for manufacturing a preparation or kit for diagnosing sarcopenia.

[0030] In addition, the present invention provides the use of a preparation for measuring the level of one or more proteins or mRNA selected from the group consisting of Asporin, PADI2, Adenosin 5'-monophosphoramidase HINT2, and HSDL2 for the diagnosis of sarcopenia. Effects of the invention

[0031] The present invention relates to compositions for the diagnosis of sarcopenia, and was completed by discovering biomarkers capable of diagnosing sarcopenia with high accuracy and sensitivity through qualitative and quantitative analysis of the proteome of an animal model of sarcopenia. In particular, it has been confirmed that the biomarkers according to the present invention have positive or negative correlations with sarcopenia-related indicators such as muscle mass, grip strength, Rotarod index, and inverted strength. Therefore, sarcopenia can be diagnosed through molecular diagnosis using the markers of the present invention, instead of conventional methods that relied on measuring muscle mass and muscle function; furthermore, it is possible to determine the severity of sarcopenia through the association between the markers and muscle function. In particular, biomarkers showing a consistent correlation with sarcopenia indicators are expected to be utilized as targets for the treatment of sarcopenia. Moreover, since sarcopenia is a disease closely related to aging, the biomarkers according to the present invention are expected to serve as a foundation for securing anti-aging treatment targets and developing aging control technologies. Brief explanation of the drawing

[0032] Figures 1a and 1b are a flowchart (Figure 1a) showing the process of protein quantification analysis of muscle tissue samples obtained from 10 ovariectomy model mice and 9 control mice, respectively, and the quantification analysis results (Figure 1b). Figures 2a and 2b show the PCA results (Figure 2a) and Volcano plot (Figure 2b) for muscle tissue proteome information of the ovariectomy model and the control group. Figure 3a is a table showing the 14 proteins that increased in the muscle tissue of the ovariectomy model compared to the control group as a result of Volcano plot analysis. Figure 3b is a table showing the 40 proteins that were reduced in the muscle tissue of the ovariectomy model compared to the control group based on the results of the Volcano plot analysis. Figures 4a to 4c show the results of gene ontology analysis of proteins increased in the muscle of an ovariectomy model. Figure 4a shows the biological processes involving the proteins, Figure 4b shows the cytological locations of the proteins, and Figure 4c shows the molecular functions of the proteins. Figure 5 shows the results of a correlation analysis between the quantitative information of muscle proteins and the muscle mass and functional characteristic index of the anterior tibialis muscle. Figure 6 shows the results of the correlation analysis between the quantitative information of muscle proteins and the Grip strength index. Figure 7 shows the results of the correlation analysis between the quantitative information of muscle proteins and the Rotarod test indicators. Figure 8 shows the results of the correlation analysis between the quantitative information of muscle proteins and the duration of the inverted grip. Specific details for implementing the invention

[0033] The present invention relates to a method for diagnosing sarcopenia, and was completed by discovering biomarkers capable of diagnosing sarcopenia with high accuracy and sensitivity through qualitative and quantitative analysis of the proteome of an animal model of sarcopenia. Specifically, the inventors performed quantitative and qualitative analyses on muscle proteins of a normal group and a sarcopenia animal model using a high-resolution mass spectrometer, and through this, selected biomarkers that showed significant differences between the groups. Furthermore, by analyzing the correlation between the said biomarkers and muscle mass, as well as the correlation with muscle function indicators such as the Grip test index, Rotarod index, and Inverted grip duration, it was confirmed that the said biomarkers have a strong correlation with muscle mass and muscle function related to sarcopenia. Therefore, the present invention is expected to be a molecular diagnostic method capable of diagnosing sarcopenia with high accuracy and efficiency, as it can replace conventional diagnostic methods that relied solely on the measurement of muscle mass and muscle function.

[0034] Accordingly, the main objective of the present invention is to provide a composition for diagnosing sarcopenia comprising, as an active ingredient, a preparation for measuring the level of one or more proteins or mRNA selected from the group consisting of Asporin, PADI2 (Protein-arginine deiminase type-2), Adenosin 5'-monophosphoramidase HINT2, and HSDL2 (hydroxysteroid dehydrogenase-like protein 2).

[0035] In the present invention, “sarcopenia” refers to a disease characterized by a decline in muscle strength and physical function due to a decrease in skeletal muscle mass. Sarcopenia is a disease that was officially recognized, having been assigned a diagnostic code as ICD-10-CM in the United States in 2016 and as KCD-8 in Korea in 2021. Patients with sarcopenia exhibit various physical functional abnormalities as muscle strength declines; for instance, their gait slows, muscle endurance decreases, and osteoporosis, falls, and fractures occur easily. Furthermore, as the blood and hormone buffering function of muscles decreases in patients with sarcopenia, their basal metabolic rate declines, making it difficult to control chronic diseases, and diabetes and cardiovascular diseases can easily worsen. While skeletal muscle is important as a motor organ, it also affects the entire body, including bones, blood vessels, the heart, and nerves; therefore, sarcopenia resulting from a decrease in skeletal muscle can lead to diseases such as fatty liver, cardiac hypertrophy, pancreatic dysfunction, diabetes, and cognitive decline.

[0036] In the present invention, sarcopenia includes sarcopenia caused by aging or menopause, as well as sarcopenia caused by other diseases. The aforementioned other diseases include, for example, obesity, infections caused by viruses or bacteria, diabetes, cancer, degenerative diseases, menopause, and degenerative diseases (such as spinal stenosis), but any disease capable of causing a decrease in muscle mass, a decrease in muscle strength, and / or a decrease in muscle function is included without limitation.

[0037] In addition, the above sarcopenia may include, but is not limited to, muscular dystrophy, useless muscle atrophy, spinal muscular atrophy, muscular dystrophy, muscle rigidity, hypotonia, muscle weakness, muscle degenerative atrophy, amyotrophic lateral sclerosis, spinal bulbar muscle atrophy, and myasthenia gravis.

[0038] In the present invention, "diagnosis" means confirming the existence or characteristics of a pathological condition, and may include confirming the presence or absence of sarcopenia, the onset of sarcopenia, the possibility of onset of sarcopenia (risk of onset), the prognosis of sarcopenia, etc.

[0039] More specifically, the term “diagnosis” as used in the present invention includes determining the susceptibility of an object to a specific disease or condition, determining whether an object currently has a specific disease or condition, determining the prognosis of an object with a specific disease or condition (e.g., identification of tumor status, determination of tumor stage, or determination of cancer responsiveness to treatment), or therametrics (e.g., monitoring the condition of an object to provide information on therapeutic efficacy).

[0040] In the present invention, “Asporin” is Aspn It refers to a protein encoded by a gene. Asporin is a cartilage extracellular protein that is a member of the leucine-rich proteoglycan family. Asporin regulates cartilage formation by inhibiting gene expression induced by TGF-β1 in cartilage, and it can bind to collagen and calcium to induce the mineralization of collagen. ASPNGenetic polymorphisms are associated with osteoarthritis and intervertebral disc disease, but their association with sarcopenia is unknown. Through proteomic analysis of muscle tissue in animal models of sarcopenia, the inventors confirmed that levels of asporin are significantly increased in sarcopenic individuals compared to normal individuals, and that there is a positive correlation, particularly with muscle mass and grip strength.

[0041] In the present invention, “PADI2 (Protein-arginine deiminase type-2)” is a protein that induces post-translational deimination of a protein by converting an arginine residue to citrulline, PADI2 It is encoded by a gene. Abnormalities in PADI2 are known to be associated with Alzheimer's disease, multiple sclerosis, glaucoma, etc., but their association with sarcopenia is unknown. Through proteomic analysis of muscle tissue in an animal model of sarcopenia, the inventors confirmed that PADI2 levels are significantly increased in sarcopenic individuals compared to normal individuals, and that there is a positive correlation, particularly with muscle functions such as inverted grip and grip strength.

[0042] In the present invention, “Adenosine 5’-monophosphoramidase HINT2 (abbreviated as HINT2)” is a protein having adenosine 5’-monophosphoramidase activity, Hint2 It is encoded by a gene. The protein hydrolyzes purine nucleotide phosphoramidates to produce AMP and NH2. HINT2 is known to be involved in steroid biosynthesis or apoptosis. Through proteomic analysis of muscle tissue in an animal model of sarcopenia, the inventors confirmed that the level of HINT2 is significantly reduced in sarcopenic individuals compared to normal individuals and has a negative correlation with muscle mass and grip strength.

[0043] In the present invention, “HSDL2 (Hydroxysteroid dehydrogenase-like protein 2)” is a protein having oxidoreductase activity, Hsdl2 It is encoded by a gene. The protein is expressed at particularly high levels in the liver, kidney, prostate, testes, and ovaries, and is also known as a major regulator of lipid metabolism; dysfunction of HSDL2 is known to be associated with gliomas or ovarian cancer. Through proteomic analysis of muscle tissue in an animal model of sarcopenia, the inventors confirmed that the level of HSDL2 is significantly reduced in sarcopenic individuals compared to normal individuals and has a negative correlation with muscle mass and grip strength.

[0044] Asporin, PADI2, Adenosin 5'-monophosphoramidase HINT2, and HSDL2 are proteins that have been identified as having the same pattern of correlation (positive or negative correlation) with two or more of the four representative diagnostic criteria for sarcopenia (muscle mass, Grip test index, Rotarod index, and Inverted grip index). In particular, all of the aforementioned proteins have been shown to have a strong correlation with the Grip test and muscle mass. Therefore, by measuring the levels of one or more of Asporin, PADI2, Adenosin 5'-monophosphoramidase HINT2, and HSDL2, not only can sarcopenia be accurately diagnosed, but the severity of sarcopenia can also be assessed based on its association with muscle mass and muscle function. For example, in patients with sarcopenia, higher protein or mRNA levels of Asporin or PADI2 are judged to indicate lower muscle mass and muscle function, which may lead to a diagnosis of high sarcopenia severity. Alternatively, in patients with sarcopenia, lower levels of protein or mRNA of Adenosin 5'-monophosphoramidase HINT2 or HSDL2 are considered to result in lower muscle mass and muscle function, which may lead to a diagnosis of high severity of sarcopenia.

[0045] 본 발명의 일 구현예에서, 상기 조성물은 Aldehyde dehydrogenase 1A1, Synaptogyrin-2, Troponin T, Pyruvate carboxylase, mitochondrial, Annexin A3, Heterogeneous nuclear ribonucleoproteins C1 / C2, Ribosome-binding protein 1, Ig gamma-3 chain C region, Ornithine aminotransferase (mitochondrial), Four and a half LIM domains protein 3, Threonine--tRNA ligase 1 (cytoplasmic), Striatin-3, Protein unc-45 homolog B, Heat shock 70 kDa protein 4, Long-chain specific acyl-CoA dehydrogenase (mitochondrial), Peptidyl-prolyl cis-trans isomerase D, Actin-binding LIM protein 1, AMP deaminase 3, Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (mitochondrial), ATP-binding cassette sub-family C member 9, Sorting nexin-3, Serine / threonine-protein phosphatase PGAM5 (mitochondrial), Eukaryotic peptide chain release factor GTP-binding subunit ERF3A, Dual specificity mitogen-activated protein kinase kinase 3, Long-chain fatty acid transport protein 1, Valacyclovir hydrolase, Myozenin-3,The agent may further include a preparation for measuring the level of one or more proteins or mRNA selected from the group consisting of [Pyruvate dehydrogenase (acetyl-transferring)] inase isozyme 4 (mitochondrial), and NADPH-cytochrome P450 reductase. The proteins (genes) are markers confirmed to be statistically significantly increased or decreased in sarcopenia animal models compared to normal individuals, and to have a positive or negative correlation with muscle mass and muscle function assessment factors (muscle mass, Grip test index, Rotarod index, and Inverted grip index).

[0046] In the present invention, Asporin, PADI2, Aldehyde dehydrogenase 1A1, Synaptogyrin-2, Troponin T, Pyruvate carboxylase, mitochondrial, Annexin A3, Heterogeneous nuclear ribonucleoproteins C1 / C2, Ribosome-binding protein 1, Ig gamma-3 chain C region, Ornithine aminotransferase (mitochondrial), Four and a half LIM domains protein 3, Threonine--tRNA ligase 1 (cytoplasmic), Striatin-3, Protein unc-45 homolog B, Heat shock 70 kDa protein 4, Long-chain specific acyl-CoA dehydrogenase (mitochondrial), and Peptidyl-prolyl cis-trans isomerase D are characterized by being increased in patients with sarcopenia compared to normal control groups. In particular, the above proteins may be further increased in patients with low muscle mass or low muscle function.

[0047] In the present invention, Adenosin 5'-monophosphoramidase HINT2, HSDL2, Actin-binding LIM protein 1, AMP deaminase 3, Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (mitochondrial), ATP-binding cassette sub-family C member 9, Sorting nexin-3, Serine / threonine-protein phosphatase PGAM5 (mitochondrial), Eukaryotic peptide chain release factor GTP-binding subunit ERF3A, Dual specificity mitogen-activated protein kinase kinase 3, Long-chain fatty acid transport protein 1, Valacyclovir hydrolase, Myozenin-3, [Pyruvate dehydrogenase (acetyl-transferring)] inase isozyme 4 (mitochondrial), and NADPH-cytochrome P450 reductase is normal control It is characterized by being reduced in patients with sarcopenia. In particular, the above proteins may be further reduced in patients with low muscle mass or low muscle function.

[0048] In this specification, “the level is increased” means that something that was not previously detected is detected, or that the amount detected is relatively higher than the normal level. For example, “the level is increased” means that the level of the experimental group is at least 1%, 2%, 3%, 4%, 5%, 10% or higher, e.g., 5%, 10%, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or higher, and / or 0.5 times, 1.1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times or higher than that of the control group. Specifically, it may mean an increase of 1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times, 4.5 to 5 times, 5 to 5.5 times, 5.5 to 6 times, 6 to 6.5 times, 6.5 to 7 times, 7 to 7.5 times, 7.5 to 8 times, 8 to 8.5 times, 8.5 to 9 times, 9 to 9.5 times, 9.5 to 10 times, or 10 times or more compared to that of the control group, but is not limited thereto. A person skilled in the art can understand the meaning of the opposite term as having the opposite meaning in accordance with the above definition.

[0049] As used herein, the term "measurement" encompasses both measuring and confirming the presence (expression) of a target substance (in the case of the present invention, the mRNA or protein of a sarcopenia biomarker gene) and measuring and confirming changes in the level of presence (expression level) of the target substance. That is, measuring the expression level of the protein means measuring whether it is expressed (i.e., measuring the presence or absence of expression), or measuring the level of qualitative and quantitative change of the protein. The measurement may be performed without limitation, including both qualitative methods (analysis) and quantitative methods. The types of qualitative and quantitative methods for measuring protein levels are well known in the art, and the experimental methods described herein are included therein. Specific methods for comparing protein levels for each method are well known in the art. Therefore, the detection of the target protein means detecting the presence of the biomarker protein, or confirming an increase (upregulation) or decrease (downregulation) in the protein level.

[0050] As used herein, the term "analysis" may preferably mean "measurement," and said qualitative analysis may mean measuring and confirming the presence of a target substance, and said quantitative analysis may mean measuring and confirming a change in the presence level (expression level) or amount of the target substance. In the present invention, analysis or measurement may be performed without limitation, including both qualitative and quantitative methods, and preferably, quantitative measurement may be performed.

[0051] If the above composition is intended to measure the level of mRNA, the agent for measuring the level of mRNA may be a primer set or probe that specifically binds to mRNA. The composition of the present invention, comprising a primer set or probe specific to the mRNA of biomarker genes according to the present invention, may further comprise an agent necessary for a known method of detecting RNA. By using a known method of detecting RNA with the composition of the present invention without limitation, the level of mRNA of the biomarkers in a subject can be measured.

[0052] In the present invention, the "primer" is a fragment that recognizes a target gene sequence and includes a forward and reverse primer pair, but preferably is a primer pair that provides analysis results having specificity and sensitivity. High specificity can be conferred when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, so that it amplifies only the target gene sequence containing the complementary primer binding site and does not induce non-specific amplification.

[0053] In the present invention, the term "probe" refers to a substance capable of specifically binding to a target substance to be detected within a sample, and means a substance capable of specifically confirming the presence of the target substance within the sample through said binding. The type of probe is not limited to substances commonly used in the industry, but preferably may be PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA, and most preferably PNA. More specifically, the probe may be a biomaterial derived from an organism or similar, or manufactured in vitro, and may be, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, DNA, and RNA; DNA may include cDNA, genomic DNA, and oligonucleotides; RNA may include genomic RNA, mRNA, and oligonucleotides; and examples of proteins may include antibodies, antigens, enzymes, peptides, etc.

[0054] Since the information (amino acid sequence, nucleic acid sequence, etc.) of the biomarker (protein or gene) according to the present invention is known, a person skilled in the art can easily design primers, probes, or antisense nucleotides that specifically bind to the gene encoding said protein based thereon. Primers or probes can be chemically synthesized using the phosphoramidite solid support synthesis method or other widely known methods and include all functional equivalents. In addition, primers or probes can be modified in various ways according to methods known in the art to the extent that they do not interfere with hybridization with mRNA. Examples of such modifications include methylation, capping, substitution with one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), and the binding of fluorescent or enzymatic labeling materials.

[0055] In cases where the composition of the present invention is intended to measure the level of protein, the agent for measuring the level of protein may be an antibody or aptamer specific to said protein.

[0056] As used herein, the term "antibody" refers to a specific protein molecule indicated for an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Furthermore, any part of a whole antibody that possesses antigen-antibody binding ability is included in the antibodies of the present invention, and all types of immunoglobulin antibodies that specifically bind to the proteins of the present invention are included. For example, this includes not only complete antibodies having two full-length light chains and two full-length heavy chains, but also functional fragments of antibody molecules, namely Fab, F(ab'), F(ab')2, and Fv, which have antigen-binding functions. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies and chimeric antibodies, as well as recombinant antibodies, provided that they can specifically bind to the proteins of the present invention.

[0057] As used herein, the term "aptamer" refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure that can specifically bind to an analyte to be detected in a sample, and can specifically confirm the presence of a target protein in a sample. The preparation of an aptamer may be carried out according to a general method for preparing aptamers, by determining and synthesizing a sequence of an oligonucleotide that has selective and high binding affinity to the target protein to be confirmed, and then modifying the 5' end or 3' end of the oligonucleotide with -SH, -COOH, -OH, or NH2 so that it can bind to the functional group of the aptamer chip, but is not limited thereto.

[0058] The composition of the present invention may additionally include a preparation necessary for a method of detecting the biomarker protein, and the expression level of a protein in a subject (or a biological sample obtained from a subject) can be measured using a known method of detecting a protein without limitation using the composition.

[0059] The composition according to the present invention may be used for the analysis of biological samples isolated from an individual requiring diagnosis. In the present invention, "biological samples" may be included without limitation as long as they are collected from a subject for whom sarcopenia is to be diagnosed or for whom the risk of onset thereof is to be predicted. For example, the biological samples may be selected from blood, whole blood, plasma, urine, saliva, tissue, muscle tissue, cells, organs, bone marrow, fine needle aspiration specimens, core needle biopsy specimens, and vacuum aspiration biopsy specimens.More specifically, the biological samples include whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, and trachea. It may be one or more selected from the group consisting of organ secretions, cells, cell extracts, and cerebrospinal fluid, but preferably, the composition according to the present invention is for non-invasive diagnosis (e.g., liquid biopsy) and may be one or more selected from the group consisting of blood, whole blood, plasma, serum, and lymph fluid. Alternatively, the biological sample may be muscle tissue, and preferably may be tissue of the tibialis anterior, extensor digitorum longus, and gastrocnemius muscles.

[0060] The biological sample may be pretreated before use for detection or diagnosis. For example, this may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, etc. The sample may be prepared to increase the detection sensitivity of protein markers, for example, the sample obtained from a subject may be pretreated using methods such as anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, sequential extraction, or gel electrophoresis.

[0061] In addition, the present invention can provide a kit for diagnosing sarcopenia comprising a composition according to the present invention.

[0062] In the present invention, the term "kit" refers to a diagnostic device or set, etc., capable of diagnosing sarcopenia by measuring the amount of mRNA or protein of biomarkers according to the present invention contained in a sample, and is not limited to any form that can measure the expression levels of said genes from a biological sample isolated from an individual. For example, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a Rapid kit, a Mass Spectrometry kit, or an MRM (Multiple Reaction Monitoring) kit, but is not limited thereto. Accordingly, the kit of the present invention may include not only antibodies that recognize target proteins as markers or primer sets and probes that recognize mRNA as markers, but also one or more other constituent compositions, solutions, or devices suitable for the analysis method. At this time, the substance for detecting the expression level of said gene, mRNA, or protein may be applied one or more times without limitation on the number of times, and there is no restriction on the order in which each substance is applied, and the application of each substance may proceed simultaneously or in stages.

[0063] In addition, the kit according to the present invention may further include an instruction manual describing a diagnostic method, etc. according to the present invention.

[0064] Another object of the present invention is to provide a method for diagnosing sarcopenia or a method for providing information for diagnosing sarcopenia, comprising the following steps:

[0065] (S1) A step of measuring the level of one or more proteins or mRNA selected from the group consisting of Asporin, PADI2, Adenosin 5'-monophosphoramidase HINT2, and HSDL2 in a biological sample isolated from a subject; and

[0066] (S2) A step of comparing the protein or mRNA level of the subject measured in the above step (S1) with a control group.

[0067] In addition, the present invention comprises: (a) measuring the level of one or more proteins or mRNA selected from the group consisting of Asporin, PADI2, Adenosin 5'-monophosphoramidase HINT2, and HSDL2 in biological samples isolated from subjects suspected of having sarcopenia and a control group; and

[0068] (b) a method for providing information for determining (or analyzing) whether a subject is highly susceptible to the development of sarcopenia, comprising the step of determining that the result indicates that the subject is highly susceptible to the development of sarcopenia, wherein the level of protein or mRNA of Asporin and / or PADI2 of the subject is increased or increased (e.g., 1.5 times, 2 times, 2.5 times, 3 times, or more) compared to the level of protein or mRNA of Asporin and / or PADI2 of the control group; and the level of protein or mRNA of Adenosin 5'-monophosphoramidase HINT2 and / or HSDL2 of the subject is decreased (e.g., 1.5 times, 2 times, 2.5 times, 3 times, or more) compared to the level of protein or mRNA of Adenosin 5'-monophosphoramidase HINT2 and / or HSDL2 of the control group.

[0069] In the present invention, the “subject” is an individual requiring diagnosis of sarcopenia, the likelihood of developing sarcopenia, muscle mass, and / or muscle function. In the present invention, the “target individual” refers to a mammal including humans, and may be selected from a group consisting of, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep, and goats, and preferably may be a human, but is not limited thereto. The subject may refer to a person who has developed sarcopenia or is suspected of developing sarcopenia, and may mean a patient who requires or is expected to receive appropriate treatment for sarcopenia, but is not limited thereto.

[0070] 본 발명의 일 구현예에서, 상기 방법은 상기 피검체로부터 분리된 생물학적 시료에서 Aldehyde dehydrogenase 1A1, Synaptogyrin-2, Troponin T, Pyruvate carboxylase, mitochondrial, Annexin A3, Heterogeneous nuclear ribonucleoproteins C1 / C2, Ribosome-binding protein 1, Ig gamma-3 chain C region, Ornithine aminotransferase (mitochondrial), Four and a half LIM domains protein 3, Threonine--tRNA ligase 1 (cytoplasmic), Striatin-3, Protein unc-45 homolog B, Heat shock 70 kDa protein 4, Long-chain specific acyl-CoA dehydrogenase (mitochondrial), Peptidyl-prolyl cis-trans isomerase D, Actin-binding LIM protein 1, AMP deaminase 3, Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (mitochondrial), ATP-binding cassette sub-family C member 9, Sorting nexin-3, Serine / threonine-protein phosphatase PGAM5 (mitochondrial), Eukaryotic peptide chain release factor GTP-binding subunit ERF3A, Dual specificity mitogen-activated protein kinase kinase 3, Long-chain fatty acid transport protein 1, Valacyclovir hydrolase, Myozenin-3,The method may further include the step of measuring the level of one or more proteins or mRNA selected from the group consisting of [Pyruvate dehydrogenase (acetyl-transferring)] inase isozyme 4 (mitochondrial), and NADPH-cytochrome P450 reductase.

[0071] In one embodiment of the present invention, the method may further include the step of diagnosing sarcopenia if the protein or mRNA level of Asporin or PADI2 measured in step (S1) is higher than that of a control group; determining that there is a risk of developing sarcopenia (high likelihood of developing sarcopenia); determining that the muscle mass is lower than that of a normal person; and / or determining that the muscle function is lower than that of a normal person.

[0072] In another embodiment of the present invention, the method may further include the step of diagnosing sarcopenia if the protein or mRNA level of Adenosin 5'-monophosphoramidase HINT2 or HSDL2 (hydroxysteroid dehydrogenase-like protein 2) measured in step (S1) is lower than that of a control group; determining that there is a risk of developing sarcopenia (high likelihood of developing sarcopenia); determining that muscle mass is lower than that of a normal person; and / or determining that muscle function is lower than that of a normal person.

[0073] In the diagnostic method according to the present invention, when analyzing receiver operating characteristics, the Area under curve may be 0.7 or higher, 0.75 or higher, 0.8 or higher, 0.85 or higher, 0.9 or higher, 0.93 or higher, 0.95 or higher, 0.96 or higher, 0.97 or higher, 0.98 or higher, or 0.99 or higher. Additionally, the diagnostic method according to the present invention may have a specificity and / or sensitivity of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 93% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher.

[0074] A detailed description regarding biomarkers, biological samples, etc., according to the present invention has been provided above and is therefore omitted.

[0075] The term "method for providing information" as used in the present invention refers to a method for providing information regarding the diagnosis of a disease, which involves analyzing biological samples of an individual or confirming increases or decreases in the levels of biomarkers according to the present invention to obtain information regarding the onset or likelihood (risk) of the disease. For example, it is a method for providing information regarding whether there is a possibility of sarcopenia developing in an individual, whether there is a relatively high possibility of sarcopenia developing, or whether sarcopenia has already developed, by measuring the levels of biomarkers according to the present invention and comparing them with a control group. Through this method, the risk of developing sarcopenia—that is, high-risk groups—can be predicted, and this can also be used as a method for providing information regarding the prevention and treatment of sarcopenia.

[0076] In the present invention, the "control" may be a biological sample isolated from a normal individual or an individual suffering from sarcopenia.

[0077] In the present invention, the method for measuring protein levels is not subject to any particular limitations as long as it is a protein measurement method known in the art, but can be measured by methods such as protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, Ouchteroni immunodiffusion method, Rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry), Western blotting, ELISA (enzyme linked immunosorbent assay), and FACS.

[0078] In the present invention, the method for measuring mRNA levels is not subject to any particular limitations as long as it is based on mRNA measurement methods known in the art, but can be measured by methods such as PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization, DNA chip, and / or RNA chip.

[0079] In addition, the present invention can provide a diagnostic device for diagnosing sarcopenia.

[0080] The measuring unit of the diagnostic device of the present invention may measure the expression level of a protein or gene using a preparation that measures the expression level of one or more proteins or genes encoding them, selected from the group consisting of biomarkers according to the present invention, such as Asporin, PADI2 (Protein-arginine deiminase type-2), Adenosin 5'-monophosphoramidase HINT2, and HSDL2 (Hydroxysteroid dehydrogenase-like protein 2), with respect to a biological sample obtained from a target individual. By confirming the degree of expression of the protein or gene using the preparation in the measuring unit, sarcopenia can be diagnosed, or a high risk of developing sarcopenia can be diagnosed.

[0081] The diagnostic device of the present invention may further include a detection unit that predicts and outputs the presence, severity, or type of sarcopenia of an individual from the degree of expression of the protein or gene obtained from the measurement unit.

[0082] In the present invention, the detection unit can diagnose sarcopenia by generating and classifying information regarding sarcopenia according to the range of the expression level of the protein or gene obtained from the measurement unit.

[0083] In addition, the present invention provides a method for treating sarcopenia comprising the following steps:

[0084] (S1) A step of measuring the level of one or more proteins or mRNA selected from the group consisting of Asporin, PADI2, Adenosin 5'-monophosphoramidase HINT2, and HSDL2 in a biological sample isolated from a subject;

[0085] (S2) A step of diagnosing the subject with sarcopenia if, by comparing the protein or mRNA level of the subject measured in step (S1) with a control group, the protein or mRNA level of the subject's Asporin or PADI2 increases compared to the control group; or the protein or mRNA level of the subject's Adenosin 5'-monophosphoramidase HINT2 or HSDL2 (hydroxysteroid dehydrogenase-like protein 2) decreases compared to the control group; and

[0086] (S3) A step of treating sarcopenia in a subject diagnosed with sarcopenia in the above step (S2).

[0087] In one embodiment of the present invention, when the protein or mRNA level of a subject measured in step (S1) is compared with a control group, if the protein or mRNA level of Asporin or PADI2 of the subject increases compared to the control group; or if the protein or mRNA level of Adenosin 5'-monophosphoramidase HINT2 or HSDL2 (hydroxysteroid dehydrogenase-like protein 2) of the subject decreases compared to the control group, the subject may be diagnosed with sarcopenia, and it may be expected that muscle mass will increase or muscle function will improve when a treatment for sarcopenia commonly used in the art is applied.

[0088] In addition, the present invention provides a method for characterizing responders to a treatment for sarcopenia (i.e., a companion diagnostic method), wherein the method comprises the following steps:

[0089] (a) a step of measuring the expression level of one or more proteins or mRNA selected from the group consisting of Asporin, PADI2, Adenosin 5'-monophosphoramidase HINT2, and HSDL2 in a biological sample isolated from a subject; and

[0090] (b) a step of determining the subject as a responder to a sarcopenia treatment by comparing the protein or mRNA level of the subject measured in step (a) with that of a control group, wherein the protein or mRNA level of the subject's Asporin or PADI2 increases compared to the control group; or the protein or mRNA level of the subject's Adenosin 5'-monophosphoramidase HINT2 or HSDL2 (hydroxysteroid dehydrogenase-like protein 2) decreases compared to the control group.

[0091] The present invention provides a method for characterizing responders for a sarcopenia treatment, characterized in that the sarcopenia treatment is one or more of Bimagrumab, Trevogrumab, Sarconeos, ARM-210, NA (cell therapy), NT-1654, AVGN7 (gene therapy), ATA 842, VB-102 (protein), Peptide of follistatin, AAV gene therapy, TEI-SARM2, Metformin, and vitamin D supplement.

[0092] In one embodiment of the present invention, the responder characterization method compares the protein or mRNA level of the subject measured in step (a) with a control group, and if the protein or mRNA level of Asporin or PADI2 of the subject increases compared to the control group; or if the protein or mRNA level of Adenosin 5'-monophosphoramidase HINT2 or HSDL2 (hydroxysteroid dehydrogenase-like protein 2) of the subject decreases compared to the control group, it can be predicted that a superior therapeutic effect will be observed when the subject is prescribed the sarcopenia treatment drug together with a sarcopenia treatment drug commonly used in the art.

[0093] The above-mentioned superior therapeutic effect includes, among other things, that the increase in muscle mass and / or improvement in muscle function appears more highly or more rapidly compared to other patients with sarcopenia. In addition, the above-mentioned treatments for sarcopenia include dietary control, protein supplementation, rehabilitation therapy, and exercise, but are not limited thereto.

[0094] In other words, the present invention provides a companion diagnostic method for the treatment of patients with sarcopenia. The term “companion diagnostics” as used in this invention refers to a biological diagnostic method used to measure a patient’s response (such as the expected degree of recovery) to a specific drug prescription or treatment. Through personalized medicine utilizing companion diagnostics, safer prescriptions are possible by reducing the risk associated with the side effects of the treatment.

[0095] In the present invention, the term “treatment” refers to any act that improves or beneficially alters the target disease and the associated metabolic abnormality symptoms, and may use methods such as chemotherapy, radiation therapy, surgical procedures, biological therapy, or antibiotic administration. For example, the treatment may be carried out through fluid therapy, medication, plasma transfusion, dietary control, exercise, rehabilitation therapy, etc.

[0096] In the present invention, the term "chemotherapy" refers to the act of using chemical substances to treat a specific disease and the entirety of the drugs used therein. Examples of the drugs include Bimagrumab, Trevogrumab, Sarconeos, ARM-210, NA (cell therapy), NT-1654, AVGN7 (gene therapy), ATA 842, VB-102 (protein), Peptide of follistatin, AAV gene therapy, TEI-SARM2, Metformin, and vitamin D supplements.

[0097] In the present invention, the surgical procedure comprises all therapeutic or diagnostic treatments involving the methodological action of the hand or the hand together with a device on the body of an individual to obtain a healing, therapeutic, or diagnostic effect.

[0098] In the present invention, the biological therapy refers to a treatment method that utilizes the human immune system directly or indirectly by using a biological preparation containing a substance derived from a living organism or a substance produced using a living organism, and the biological preparation includes vaccines, allergens, antigens, hormones, cytokines, enzymes, blood and plasma, immune serum, monoclonal antibodies, fermented products, antitoxins, and laboratory diagnostic agents, the potency and stability of which cannot be evaluated by physical and chemical tests alone.

[0099] In particular, the biomarkers according to the present invention are proteins that have a strong correlation with sarcopenia and have potential as therapeutic targets. Therefore, for the treatment of sarcopenia, expression inhibitors or activity inhibitors of Asporin and / or PADI2 may be used, or expression promoters or activators of Adenosin 5'-monophosphoramidase HINT2 and / or HSDL2 may be used.

[0101] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0103] [Example]

[0104] Example 1. Preparation of an animal model of sarcopenia using ovariectomy

[0105] In this study, an animal model of sarcopenia was constructed by inducing sarcopenia in mice through ovary-sectomization. Specifically, 7-week-old female C57BL / 6 mice were purchased, stabilized under specific pathogen-free conditions for 2 weeks, and underwent either bilateral ovariectomy or sham surgery at 9 weeks of age. Two months after surgery, the mice were sacrificed, and blood, tibialis anterior, extensor digitorum longus, and gastrocnemius muscle tissues were collected; among these, quantitative proteomic analysis was performed on the tibialis anterior tissue. The characteristics of the mice two months after surgery for the proteomic study are shown in Table 1 below.

[0106] 2 months after surgery 　 Sham (n = 9) OVX (n = 10) Δ change P Body weight (g) 23.0 ± 1.2 30.1 ± 2.5 30.9% <0.001 BMD (g / cm3) 0.074 ± 0.003 0.068 ± 0.003 -9.5% <0.001 Fat mass (g) 1.08 ± 0.45 4.75 ± 1.12 339.8% <0.001 Lean mass (g) 18.8 ± 1.0 23.3 ± 1.6 23.9% <0.001 Sarcopenic index (%) Soleus 0.037 ± 0.004 0.030 ± 0.007 -18.9% 0.027 Tibialis anterior 0.173 ± 0.029 0.129 ± 0.013 -25.4% <0.001 Extensor digitorum longus 0.041 ± 0.007 0.026 ± 0.012 -36.6% 0.005 Gastrocnemius 0.532 ± 0.035 0.400 ± 0.020 -24.8% <0.001 Grip strength (g / g body weight) 6.91 ± 0.54 5.28 ± 0.46 -23.6% <0.001 Latency time to fall(s) 85.3 ± 24.5 54.9 ± 8.7 -35.6% 0.006 Grid hanging time (s) 66.8 ± 63.1 8.6 ± 3.3 -87.1% 0.024

[0108] Example 2. Sample preparation for proteomic analysis

[0109] Tibialis anterior muscle tissue was lysed in 100 μL of lysis buffer containing a mixture of 5% sodium dodecyl sulfate and 50 mM triethylammonium bicarbonate (pH 7.55, Thermo Fisher Scientific). Lysis was performed using the Adaptive Focused Acoustics technique, and samples were treated with S-trap-based trypsin digestion according to the manufacturer's instructions, with the exception of the trypsin / LysC mixture (Promega, Madison, WI, USA). Dried peptides were reconstituted with 0.1% formic acid, and absorbance at 205 nm was measured using a NanoDrop One spectrophotometer (ThermoFisher Scientific) to determine peptide concentration. 4 μg of peptide from each sample was allocated for individual analysis and dried. All remaining peptides were combined to generate a spectroscopic library. .

[0111] Example 3. Mass spectrometry analysis for quantitative proteomics

[0112] Individual muscle peptide samples were analyzed using a SCIEX TripleTOF 5600+ system mass spectrometer with the following LC-MS / MS setup: for LC separation, an Eksigent microtrap cartridge (ChromXP C18CL, 5 μm, 120 Å) was used as the trap column and an Eksigent column (C18-CL, 0.3 x 150 mm, particle size 3 μm, pore size 120 Å) was used as the analytical column, and the column temperature was maintained at 40 ℃. TMnanoLC 425 (Eksigent, Dublin, CA) was used. Samples were loaded onto a trap column at a flow rate of 10 µl / min using 100% eluent A: 0.1% formic acid. After 10 minutes, peptides were separated in 87-minute intervals at 5 µl / min using eluent A and eluent B: 0.1% formic acid in 100% acetonitrile (eluent B was separated by 3 to 25% over 68 minutes, 25 to 35% over 5 minutes, 35 to 80% over 2 minutes, maintained at 80% for 3 minutes, maintained at 80% for 1 minute, and maintained at 3% for 8 minutes). Mass spectrometry was operated using 100 variable windows in Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH) mode according to the SCIEX technical note. The SWATH parameters were set as follows: lower limit m / z 400; upper limit m / z 1250; window overlap (Da) 1.0; and CES was set to 5 for small windows, 8 for large windows, and 10 for the largest windows. MS2 spectra were collected in the 100–1500 m / z range for 2.5 ms in high-sensitivity mode, and the total cycle time was 2.8 seconds. The analysis process of muscle proteins using mass spectrometry is shown in Fig. 1a.

[0114] Example 4. SWATH Data Analysis

[0115] SWATH-MS data for individual samples in-houseProcessing was performed using DIA-NN 1.7.10 with the urine proteome spectroscopic library. Spectra were retrieved with default settings, but the m / z range was 400–1250 for precursors and 100–1500 for fragment ions. Protein quantification values ​​were obtained using the MaxLFQ algorithm (PMID: 24942700) implemented in the Diann R package (https: / / github.com / vdemichev / diann-rpackage). Protein intensity values ​​were filtered for precursor and protein groups passing through q-values ​​of 0.01 or higher.

[0117] Example 5. Statistical Analysis

[0118] The free software Perseus (version 1.6.14.0) was used for differential analysis of protein intensity between samples. Normalized protein intensity values ​​were converted to a log2 scale. Each sample was grouped by type, requiring at least 90% from at least one group. Missing values ​​were replaced with random numbers drawn from a normal distribution using default parameters (width: 0.3, downshift: 1.8). T-tests were performed using the Benjamini-Hochberg FDR (0.05 cutoff) to identify statistically significant differences between samples. Visualization of volcano plots and box plots was performed using InstantClue software.

[0120] [Experimental Example]

[0121] Experimental Example 1. SWATH analysis for the discovery of biomarkers associated with sarcopenia

[0122] To select candidate biomarkers associated with sarcopenia, proteins were extracted from mouse muscle tissues of 10 ovariectomized models and 9 control mouse muscle tissues using the method described in the above example, and qualitative and quantitative protein analysis was performed using SWATH-MS. Quantitative and qualitative information were extracted from the spectrum of the obtained peptides using DIA-NN, and a total of 2,086 proteins were quantified as shown in Fig. 1b.

[0124] Experimental Example 2. Confirmation of characteristics of muscle proteome in a sarcopenia model

[0125] After removing one outlier from the control group and one from the ovariectomy group and performing Principal component analysis, it was confirmed that the ovariectomy model group and the control group were well separated, as shown in Figure 2a. In addition, a volcano plot was performed using the fold change and P value for the quantitative values ​​of proteins relative to the control group, and as shown in Figure 2b, it was confirmed that 14 proteins increased and 30 proteins decreased in the muscle tissue of the ovariectomy model.

[0126] In the Volcano plot analysis, 14 proteins that increased in the muscle tissue of the ovariectomy model compared to the control group are shown in Figure 3a. Pregnacy zone protein was found to increase threefold in the ovariectomy model compared to the control group, and Plasma protease C1 inhibitor was also found to increase 2.6fold.

[0127] Figure 3b shows the 40 proteins that decreased in the muscle tissue of the ovariectomy model compared to the control group in the Volcano plot analysis. Myosin-binding protein C, fast-type was found to decrease by 0.38 times in the muscle of the ovariectomy model compared to the control group, and platelet factor 4 was found to decrease by 0.48 times.

[0128] Gene ontology analysis of proteins increased in the muscle tissue of the ovariectomy model compared to the control group, based on Volcano plot analysis, confirmed that these proteins are involved in muscle contraction and the regulation of injured axons (Fig. 4a). Furthermore, analysis of the cellular compartments of these proteins revealed that most were located in cytological positions associated with contractile muscle and supramolecular fibers (Fig. 4b). From these results, it can be expected that sarcopenia caused by ovariectomy leads to an increase in proteins associated with the tibialis anterior muscle rather than other muscles, thereby causing sarcopenia and functional decline in muscle mass. Additionally, molecular functions of these proteins were identified as possessing actin binding and action filament biding capabilities (Fig. 4c).

[0130] Experimental Example 3. Discovery of muscle proteins showing a correlation with muscle mass

[0131] The Pearson correlation coefficient was determined by analyzing the correlation between the quantitative information of 1,527 proteins, whose quantification was confirmed by performing missing value replacement, and the amount of anterior tibialis muscle. As a result, as shown in Figure 5, the proteins Aldh1a1, Aspn, Syngr2, Tnnt3, and Pc were found to have a positive correlation with the amount of muscle. In addition, the proteins Ablim1, Ampd3, Ech1, Hint2, and Hsdl2 were found to have a negative correlation with the amount of muscle.

[0133] Experimental Example 4. Discovery of muscle proteins showing a correlation with the Grip test index

[0134] The Pearson correlation coefficient was determined by analyzing the correlation between the Grip test index, an index related to muscle function, and the quantities of muscle proteins. As a result, as shown in Figure 6, Anxa3, Hnrnpc, Rrbp1, Padi2, and Aspn showed a positive relationship with muscle strength. Furthermore, Hsdl2, Fam162a, Mcu, Hint2, and Bphl were found to have a negative correlation with muscle strength. These results suggest that muscle strength can be expressed through the quantitative relationship of intramuscular proteins.

[0136] Experimental Example 5. Discovery of muscle proteins correlated with the Rotarod index

[0137] The Pearson correlation coefficient was determined by analyzing the correlation between quantitative information on muscle protein and the indicators of the Rotarod test, another form of muscle function indicator. As a result, as shown in Figure 7, Ighg3, Oat, Fhl3, Tars1, and Strn3 proteins were found to have a positive correlation with the Rotarod test results. Abcc9, Snx3, Pgam5, Gspt1, and Map2k3 showed a negative correlation with the Rotarod test results.

[0139] Experimental Example 6. Discovery of muscle proteins correlated with inverted grip duration

[0140] The Pearson correlation coefficient was determined by analyzing the correlation between inverted grip duration, another indicator of muscle function, and quantitative information of muscle proteins. As a result, as shown in Figure 8, Unc45b, Hspa4, Acadl, Padi2, and Ppid showed a very strong positive relationship with inverted grip duration. Unc45b showed a Pearson correlation index of 0.859 with inverted grip duration. Additionally, the quantitative information of Slc27a1, Bphl, Myoz3, Pdk4, and Por proteins was found to have a strong negative correlation with inverted grip duration.

[0142] 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.

Claims

Claim 1 A composition for diagnosing sarcopenia comprising, as an active ingredient, a preparation for measuring the protein or mRNA levels of PADI2 (Protein-arginine deiminase type-2). Claim 2 A composition for diagnosing sarcopenia according to claim 1, further comprising a preparation for measuring the level of one or more proteins or mRNA selected from the group consisting of Asporin, Adenosin 5'-monophosphoramidase HINT2, and HSDL2 (Hydroxysteroid dehydrogenase-like protein 2). Claim 3 A composition for diagnosing sarcopenia, characterized in that, in paragraph 2, the protein or mRNA levels of the above-mentioned Asporin or PADI2 are increased in patients with sarcopenia compared to normal individuals. Claim 4 A composition for diagnosing sarcopenia according to claim 2, characterized in that the protein or mRNA levels of the Adenosin 5'-monophosphoramidase HINT2 or HSDL2 are reduced in patients with sarcopenia compared to normal individuals. Claim 5 제2항에 있어서,상기 조성물은 Aldehyde dehydrogenase 1A1, Synaptogyrin-2, Troponin T, Pyruvate carboxylase, mitochondrial, Annexin A3, Heterogeneous nuclear ribonucleoproteins C1 / C2, Ribosome-binding protein 1, Ig gamma-3 chain C region, Ornithine aminotransferase (mitochondrial), Four and a half LIM domains protein 3, Threonine--tRNA ligase 1 (cytoplasmic), Striatin-3, Protein unc-45 homolog B, Heat shock 70 kDa protein 4, Long-chain specific acyl-CoA dehydrogenase (mitochondrial), Peptidyl-prolyl cis-trans isomerase D, Actin-binding LIM protein 1, AMP deaminase 3, Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (mitochondrial), ATP-binding cassette sub-family C member 9, Sorting nexin-3, Serine / threonine-protein phosphatase PGAM5 (mitochondrial), Eukaryotic peptide chain release factor GTP-binding subunit ERF3A, Dual specificity mitogen-activated protein kinase kinase 3, Long-chain fatty acid transport protein 1, Valacyclovir hydrolase, Myozenin-3,A composition for diagnosing sarcopenia, characterized by further comprising a preparation for measuring the level of one or more proteins or mRNA selected from the group consisting of [Pyruvate dehydrogenase (acetyl-transferring)] inase isozyme 4 (mitochondrial), and NADPH-cytochrome P450 reductase. Claim 6 A composition for diagnosing sarcopenia, characterized in that, in claim 1, the preparation for measuring the protein level is an antibody or aptamer specific to the protein. Claim 7 A composition for diagnosing sarcopenia according to claim 1, wherein the preparation for measuring the mRNA expression level is a primer set or probe that specifically binds to the mRNA. Claim 8 A composition for diagnosing sarcopenia according to claim 1, wherein the protein or mRNA level is a level in one or more selected from the group consisting of blood, whole blood, plasma, lymph fluid, urine, saliva, tissue, muscle tissue, cell, organ, bone marrow, fine needle aspiration specimen, core needle biopsy specimen, and vacuum aspiration biopsy specimen. Claim 9 A kit for diagnosing sarcopenia comprising a composition of any one of claims 1 to 8. Claim 10 A method for providing information for the diagnosis of sarcopenia comprising the following steps: (S1) measuring the protein or mRNA level of PADI2 in a biological sample isolated from a subject; and (S2) comparing the protein or mRNA level of the subject measured in step (S1) with a control group. Claim 11 A method for providing information for the diagnosis of sarcopenia, wherein the method further comprises the step of measuring the level of one or more proteins or mRNA selected from the group consisting of Asporin, Adenosin 5'-monophosphoramidase HINT2, and HSDL2 in step (S1). Claim 12 A method for providing information for the diagnosis of sarcopenia, wherein the method further comprises the step of determining that if the protein or mRNA level of Asporin or PADI2 measured in step (S1) is higher than that of a control group, the person has sarcopenia or is at risk of developing sarcopenia. Claim 13 A method for providing information for the diagnosis of sarcopenia, wherein the method further comprises the step of determining that a person has sarcopenia or is at risk of developing sarcopenia if the protein or mRNA level of Adenosin 5'-monophosphoramidase HINT2 or HSDL2 (hydroxysteroid dehydrogenase-like protein 2) measured in step (S1) is lower than that of a control group. Claim 14 제11항에 있어서,상기 방법은 상기 피검체로부터 분리된 생물학적 시료에서 Aldehyde dehydrogenase 1A1, Synaptogyrin-2, Troponin T, Pyruvate carboxylase, mitochondrial, Annexin A3, Heterogeneous nuclear ribonucleoproteins C1 / C2, Ribosome-binding protein 1, Ig gamma-3 chain C region, Ornithine aminotransferase (mitochondrial), Four and a half LIM domains protein 3, Threonine--tRNA ligase 1 (cytoplasmic), Striatin-3, Protein unc-45 homolog B, Heat shock 70 kDa protein 4, Long-chain specific acyl-CoA dehydrogenase (mitochondrial), Peptidyl-prolyl cis-trans isomerase D, Actin-binding LIM protein 1, AMP deaminase 3, Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (mitochondrial), ATP-binding cassette sub-family C member 9, Sorting nexin-3, Serine / threonine-protein phosphatase PGAM5 (mitochondrial), Eukaryotic peptide chain release factor GTP-binding subunit ERF3A, Dual specificity mitogen-activated protein kinase kinase 3, Long-chain fatty acid transport protein 1, Valacyclovir hydrolase, Myozenin-3,A method for providing information for the diagnosis of sarcopenia, further comprising the step of measuring the level of one or more proteins or mRNA selected from the group consisting of [Pyruvate dehydrogenase (acetyl-transferring)] inase isozyme 4 (mitochondrial), and NADPH-cytochrome P450 reductase. Claim 15 A method for providing information for the diagnosis of sarcopenia, wherein the biological sample is one or more selected from the group consisting of blood, whole blood, plasma, lymph fluid, urine, saliva, tissue, muscle tissue, cell, organ, bone marrow, fine needle aspiration specimen, core needle biopsy specimen, and vacuum aspiration biopsy specimen. Claim 16 A method for providing information for the diagnosis of sarcopenia according to claim 10, wherein the protein level is measured by one or more methods selected from the group consisting of protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, Ouchteroni immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry), Western blotting, ELISA (enzyme linked immunosorbent assay), and FACS. Claim 17 A method for providing information for the diagnosis of sarcopenia, characterized in that, in claim 10, the mRNA level is measured by one or more methods selected from the group consisting of PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization, DNA chip, and RNA chip.

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