WO2023127998A1 - Composition for predicting occurrence of sarcopenia in hip fracture patients - Google Patents

Abstract

The present invention relates to a composition for predicting occurrence of sarcopenia in hip fracture patients. More specifically, by comprising an agent that measures the expression level of a gene selected from the group consisting of RUNX 1, NGFR, CH3L1, BCL3, PLA2G2A, MYBPH, TEP1, SEMA6B, CSPG4, ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP, the present invention allows rehabilitation treatment to be more effective for hip fracture patients.

Description

Composition for predicting the onset of sarcopenia in hip fracture patients

The present invention relates to a composition for predicting the onset of sarcopenia in a hip fracture individual and a method for providing information for predicting whether or not the onset of sarcopenia occurs in a hip fracture individual.

In the case of the elderly, the prevalence of many chronic diseases such as osteoporosis and sarcopenia is high. Osteoporosis-related studies in the elderly population have reported clinical results and established treatment guidelines over the past decades, but recently interest in sarcopenia and related research has increased.

Sarcopenia appears primarily in metabolic diseases such as diabetes, obesity, and cachexia, and in some specific diseases, including chronic renal failure, congestive heart failure, and chronic obstructive pulmonary disease. However, sarcopenia is significantly associated with osteoporosis in the elderly population. Several studies have shown that sarcopenia and osteoporosis share common risk factors and biological pathways, and that osteopenia is associated with severe physical disability and represents a serious threat in old age. The combination of these two diseases has been described as a "hazardous duet" that exacerbates the negative health outcomes and adds a tendency to fall from sarcopenia to the bone fragility of people with osteoporosis. The combined effects of sarcopenia and osteoporosis in the elderly represents a serious problem for them.

The present inventors completed the present invention with the support of the National Research Foundation of Korea (NRF) funded by the Korean government (Ministry of Education, Science and Technology, MEST) (No. NRF-2019R1F1A1059208).

[Assignment identification number] 1711145790

[Assignment number] 2019R1F1A1059208

[Name of Department] Ministry of Science and ICT

[Name of project management (professional) institution] National Research Foundation of Korea

[Research project name] Individual basic research (Ministry of Science and ICT) (R&D)

[Research title] Establishment of multi-omics and bio-big data precision medical foundation to overcome osteoarthritis in the elderly

[Contribution rate] 1/1

[Name of project performing institution] Gyeongsang National University Hospital

[Research Period] 2021-03-01~2022-02-28

An object of the present invention is to provide a composition for predicting the onset of sarcopenia in a hip fracture subject.

An object of the present invention is to provide a kit for predicting the onset of sarcopenia in a hip fracture subject.

An object of the present invention is to provide a method for providing information for predicting the onset of sarcopenia in a hip fracture subject.

1. RUNX 1 (RUNX family transcription factor 1), NGFR (nerve growth factor receptor), CH3L1 (Chitinase-3-like protein 1), BCL3 (B cell lymphoma 3), PLA2G2A (Phospholipase A2 Group IIA), MYBPH (myosin binding protein H), TEP1 (telomerase associated protein 1), SEMA6B (semaphorin 6B), CSPG4 (chondroitin sulfate proteoglycan 4), ACSL5 (acyl-CoA synthetase long chain family member 5), SLC25A3 (solute carrier family 25 member 3), NDUFB5 (NADH: ubiquinone oxidoreductase subunit B5), CYC1 (cytochrome c1), ACAT1 (acetyl-CoA acetyltransferase 1), and TCAP (titin-cap), comprising an agent for measuring the expression level of at least one gene selected from the group consisting of A composition for predicting the occurrence of sarcopenia in a hip fracture subject.

2. The composition according to 1 above, wherein the agent is at least one selected from the group consisting of primers, probes, antibodies, aptamers, DNA, RNA, proteins and polypeptides, the composition for predicting the onset of sarcopenia in hip fracture individuals.

3. The composition for predicting the onset of sarcopenia in a hip fracture subject according to 1 above, wherein the hip fracture subject is accompanied by osteoporosis.

4. A kit for predicting the onset of sarcopenia in a hip fracture subject comprising the composition of any one of 1 to 3 above.

5. RUNX 1 (RUNX family transcription factor 1), NGFR (nerve growth factor receptor), CH3L1 (Chitinase-3-like protein 1), BCL3 (B cell lymphoma 3), PLA2G2A ( Phospholipase A2 Group IIA), myosin binding protein H (MYBPH), telomerase associated protein 1 (TEP1), semaphorin 6B (SEMA6B), chondroitin sulfate proteoglycan 4 (CSPG4), acyl-CoA synthetase long chain family member 5 (ACSL5), SLC25A3 (solute carrier family 25 member 3), NDUFB5 (NADH: ubiquinone oxidoreductase subunit B5), CYC1 (cytochrome c1), ACAT1 (acetyl-CoA acetyltransferase 1) and TCAP (titin-cap) of at least one gene selected from the group consisting of A method of providing information for predicting the onset of sarcopenia in a hip fracture subject, comprising the step of comparing the expression level with the expression level in a control group.

6. In the above 5, if the expression level of at least one gene selected from the group consisting of RUNX 1, NGFR, CH3L1, BCL3 and PLA2G2A is higher than the expression level in the control group, information that the possibility of developing sarcopenia compared to the control group is high A method for providing information, further comprising the step of providing.

7. In the above 5, if the expression level of at least one gene selected from the group consisting of MYBPH, TEP1, SEMA6B and CSPG4 is higher than the expression level in the control group, providing information that the possibility of developing sarcopenia compared to the control group is high Further comprising, information providing method.

8. In the above 5, if the expression level of at least one gene selected from the group consisting of ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP is lower than the expression level in the control group, sarcopenia compared to the control group is more likely to develop An information providing method, further comprising providing information.

9. In the above 5, selected from the group consisting of RUNX 1, NGFR, CH3L1, BCL3, PLA2G2A, MYBPH, TEP1, SEMA6B, CSPG4, ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP in the sample isolated from the subject Further comprising the step of measuring the expression level of at least one gene, information providing method.

10. The information providing method according to 5 above, wherein the sample is one selected from the group consisting of muscle cells, muscle tissue, plasma, serum and urine.

11. The information providing method according to 5 above, wherein the hip fracture subject is accompanied by osteoporosis.

The composition and information providing method of the present invention can help to more effectively perform rehabilitation treatment by predicting the onset of sarcopenia in a hip fracture subject.

1 is a schematic diagram showing the process of selecting a test subject.

FIG. 2A shows a differentially expressed gene (DEG) heatmap that clearly separates sarcopenia from normal patients, and FIG. 2B is a swarm plot of known genes in each expression group. is shown.

Figure 3 is a DEG classification by Kegg Brite classification, Figure 3A shows a network-based Keg Bright classification (the size of a circle represents the number of corresponding genes, and dots represent genes corresponding to the corresponding class), Figure 3B shows It represents the genetic members of the "Exosome" and "Enzyme" classes found mostly in DEGs.

Figure 4 shows the one-step neighbor of DEGs based on the HuRI network (among the genes circled, RUNX1, MRPL45, TCAP, TEP1, SLC25A3, NDUFB5, ACSL5, HPN are DEGs, BAG3, SNRPC, VPS37C, HGS , BAG4, NFKBID, KRTAP19-7, RHOXF2, C19orf54, BLZF1, IKZF3, PFDN5, EMD, RHBDD2, SLC10A1, TMEM14B, AQP6, SLC10A6, RETREG3, SLC35C2, BNIP3 are known network hubs of one step neighbors of the above DEGs. others indicate non-hub interactors of DEGs).

5 shows the qPCR verification results.

Hereinafter, the present invention will be described.

The present invention relates to a composition for predicting the onset of sarcopenia in a hip fracture subject.

The composition is RUNX 1 (RUNX family transcription factor 1), NGFR (nerve growth factor receptor), CH3L1 (Chitinase-3-like protein 1), BCL3 (B cell lymphoma 3), PLA2G2A (Phospholipase A2 Group IIA), MYBPH ( myosin binding protein H), TEP1 (telomerase associated protein 1), SEMA6B (semaphorin 6B), CSPG4 (chondroitin sulfate proteoglycan 4), ACSL5 (acyl-CoA synthetase long chain family member 5), SLC25A3 (solute carrier family 25 member 3) , NDUFB5 (NADH: ubiquinone oxidoreductase subunit B5), CYC1 (cytochrome c1), ACAT1 (acetyl-CoA acetyltransferase 1), and TCAP (titin-cap), including an agent for measuring the expression level of at least one gene selected from the group consisting of do.

Hip fracture refers to a fracture that occurs mainly by direct impact on the outside of the hip joint. Hip fractures are mostly caused by high-energy trauma such as falls or traffic accidents in young people, and in elderly patients due to weakened bone quality, most of them are caused by low-energy injuries such as simple falls.

Sarcopenia refers to a disease in which the amount, strength, and function of muscles decrease due to various reasons such as aging. Sarcopenia may be defined by Asia Working Group for Sarcopenia (AWGS) criteria.

Patients with hip fractures accompanied by sarcopenia are known to have a poor prognosis even after surgery.

The subject may be an animal, and specifically may be a human, cow, horse, rabbit, dog, monkey, or cat, but is not limited thereto.

The subject may be a hip fracture patient with osteoporosis. The subject may be an elderly hip fracture patient, specifically, a hip fracture patient aged 65 years or older. The subject may be a female hip fracture patient.

At least one selected from the group consisting of RUNX 1, NGFR, CH3L1, BCL3, PLA2G2A, MYBPH, TEP1, SEMA6B, CSPG4, ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP measured in samples isolated from hip fracture patients The correlation between the expression level of the gene and the occurrence of sarcopenia was confirmed. Therefore, the expression level of each of the above genes can be used to predict the occurrence of sarcopenia in a hip fracture subject.

It is known that the prognosis is poor when hip fracture individuals are accompanied by sarcopenia. Therefore, the composition of the present invention can be used to predict the onset of sarcopenia in a hip fracture subject, and thus can help predict the prognosis of a hip fracture subject.

The expression level may be an mRNA expression level of the gene or an expression level of a protein encoded by the gene.

The agent for measuring the expression level is limited to a substance capable of detecting RUNX 1, NGFR, CH3L1, BCL3, PLA2G2A, MYBPH, TEP1, SEMA6B, CSPG4, ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP mRNA or its protein However, it may be at least one selected from the group consisting of primers, probes, antibodies, aptamers, DNA, RNA, proteins, and polypeptides.

The term “prediction” means to guess in advance about a medical outcome.

The present invention provides a kit for predicting the onset of sarcopenia in a hip fracture subject comprising the above-described composition.

The kit measures the expression level of at least one gene selected from the group consisting of RUNX 1, NGFR, CH3L1, BCL3, PLA2G2A, MYBPH, TEP1, SEMA6B, CSPG4, ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP described above contains the formulation.

The kit may include essential components required to perform ELISA in order to implement various ELISA methods such as sandwich ELISA. In addition, the kit may include essential components such as a fluorescent antibody necessary for performing flow cytometry.

The type of kit may be any kit known to those skilled in the art, and may be, for example, an immunochromatography strip kit, a Luminex assay kit, a protein microarray kit, an ELISA kit, a flow cytometry kit, or an immunological dot kit.

The kit may be a kit for implementing Western blot, immunoprecipitation assay, complement fixation assay, flow cytometry, or protein chip, and may further include additional components suitable for each assay method.

The present invention provides a method for providing information for predicting whether sarcopenia occurs in a hip fracture subject.

The information providing method is RUNX 1 (RUNX family transcription factor 1), NGFR (nerve growth factor receptor), CH3L1 (Chitinase-3-like protein 1), BCL3 (B cell lymphoma 3) measured in samples isolated from the subject , PLA2G2A (Phospholipase A2 Group IIA), MYBPH (myosin binding protein H), TEP1 (telomerase associated protein 1), SEMA6B (semaphorin 6B), CSPG4 (chondroitin sulfate proteoglycan 4), ACSL5 (acyl-CoAthetase long chain synthetase family member 5 ), at least one selected from the group consisting of SLC25A3 (solute carrier family 25 member 3), NDUFB5 (NADH: ubiquinone oxidoreductase subunit B5), CYC1 (cytochrome c1), ACAT1 (acetyl-CoA acetyltransferase 1), and TCAP (titin-cap) Comparing the expression level of the gene of with the expression level in the control group.

Hip fracture, sarcopenia, subject, expression level, and prediction may be within the above range, but are not limited thereto.

The subject may be an individual with a fractured hip joint. The subject may be an individual who has experienced hip fracture surgery or is scheduled to undergo hip fracture surgery.

A sample isolated from a subject may be selected from the group consisting of muscle cells, muscle tissue, plasma, serum, and urine.

Controls can be normal individuals or hip fracture patients. A control group may be a hip fracture patient without sarcopenia. A control group may be a hip fracture patient with osteoporosis without sarcopenia.

At least one selected from the group consisting of RUNX 1, NGFR, CH3L1, BCL3, PLA2G2A, MYBPH, TEP1, SEMA6B, CSPG4, ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP measured in samples isolated from hip fracture patients The correlation between the expression level of the gene and the occurrence of sarcopenia was confirmed. Therefore, the expression level can be used to predict the onset of sarcopenia in hip fracture individuals.

The method of providing information is that if the expression level of at least one gene selected from the group consisting of RUNX 1, NGFR, CH3L1, BCL3 and PLA2G2A is higher than the expression level in the control group, sarcopenia compared to the control group is likely to occur. To provide information Further steps may be included.

Specifically, the inventors confirmed that the expression level of at least one gene selected from the group consisting of RUNX 1, NGFR, CH3L1, BCL3 and PLA2G2A measured in a sample isolated from the subject correlated positively with the possibility of developing sarcopenia did Therefore, if the expression level of at least one gene selected from the group consisting of RUNX 1, NGFR, CH3L1, BCL3 and PLA2G2A measured in a sample isolated from the subject is higher than the expression level in the control group, the possibility of developing sarcopenia compared to the control group is high. can provide information.

The information providing method further includes the step of providing information that, when the expression level of at least one gene selected from the group consisting of MYBPH, TEP1, SEMA6B, and CSPG4 is higher than that in the control group, the possibility of developing sarcopenia is high compared to the control group. can include

Specifically, the present inventors confirmed that the expression level of at least one gene selected from the group consisting of MYBPH, TEP1, SEMA6B, and CSPG4 measured in a sample isolated from a subject has a positive correlation with the possibility of developing sarcopenia. Therefore, if the expression level of at least one gene selected from the group consisting of MYBPH, TEP1, SEMA6B and CSPG4 measured in a sample isolated from the subject is higher than the expression level in the control group, it provides information that the possibility of developing sarcopenia compared to the control group is high. can do.

The method of providing information is that if the expression level of at least one gene selected from the group consisting of ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP is lower than the expression level in the control group, the possibility of developing sarcopenia compared to the control group is high. The step of providing may be further included.

Specifically, the present inventors found that the expression level of at least one gene selected from the group consisting of ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP measured in a sample isolated from a subject has a negative correlation with the possibility of developing sarcopenia Confirmed. Therefore, if the expression level of at least one gene selected from the group consisting of ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP measured in a sample isolated from the subject is lower than the expression level in the control group, there is a possibility of developing sarcopenia compared to the control group. It can provide information that will be high.

The information providing method is at least one selected from the group consisting of RUNX 1, NGFR, CH3L1, BCL3, PLA2G2A, MYBPH, TEP1, SEMA6B, CSPG4, ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP in a sample isolated from the subject A step of measuring the expression level of the gene may be further included.

The step of measuring the expression level of the gene may be performed by treating the sample with an agent for measuring the mRNA expression level of the gene to be measured or the expression level of the protein encoded by the gene.

Reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Real-time RT-PCR), RNase protection assay ( RPA; RNase protection assay), Northern blotting, and DNA chips may be used, but are not limited thereto.

Analysis methods for measuring protein expression include western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, and Ouchterlony immunodiffusion method. , Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FACS), protein chip, etc. , but is not limited thereto.

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

Example

Experiment method

1. Test subject

The patient group included subjects aged 65 years or older who underwent hip fracture surgery. Among 323 patients with hip fracture (HF) confirmed from May 2017 to December 2019, 162 patients with hip fracture (excluding high energy trauma and non-osteoporosis) (90 patients with non-sarcopenia and 72 patients with sarcopenia) were included in the final analysis. For RNA sequencing, each patient with the top 10% of hand grip strength (HGS) value was set as the control group, and the bottom 10% was set as the patient group, except for patients with poor tissue quality, the sarcopenia group and the proximal muscle group. Six and five patients were selected as the saponification group, respectively. For qPCR verification, each patient with HGS value in the top 20% and bottom 20% was set as a control group and patient group. Excluding patients with poor tissue quality, 12 patients and 12 patients in the sarcopenia group and the non-sarcopenia group, respectively, were selected. Four patients were selected (Fig. 1).

The experimental protocol was approved by the Institutional Review Board (IRB) of Gyeongsang National University Hospital (IRB number: 2019-02-013).

2. Preparation of muscle samples

Muscle samples were collected through a 5×5×3 mm gluteus maximus obtained from a surgical opening. Tissues were stored at -80°C at Tissue-Tek (Miles, Elkhart, IN, USA).

3. Diagnosis of osteosarcomenia

Body composition was measured by whole-body DXA (DPX-NT; GE Medical Systems Lunar, Madison, WI, USA). Bone mineral content, fat mass, and lean soft tissue mass (lean mass) were separately measured for each body part including arms and legs. The lean soft tissue mass of the arms and legs was approximately equal to the skeletal muscle mass. Since absolute muscle mass is correlated with height, the skeletal muscle mass index (SMI) was calculated with the following formula: lean mass [kg]/height ² [m ² ], which is It is directly similar to body mass index (BMI) (BMI: weight [kg]/height ² [m ² ]). Arm SMI was defined as “arm lean mass [kg]/height ² [m ² ]”. Leg SMI was defined as “lean mass [kg]/height ² [m ² ]”. Limb SMI (Appendicular SMI) was defined as the sum of arm SMI and leg SMI.

Muscle strength was evaluated by grip strength using an analogue dynamometer (TK 5001 Grip-A, Takei, Tokyo, Japan). In a sitting position, with the shoulder attached to the body, the elbow bent at 90°, and the wrist maintaining a neutral posture (0°), the grip force giving the maximum force was measured. Sarcopenia is defined as low muscle strength (grip strength less than 18 kg for women and less than 28 kg for men) and reduced muscle mass (SMI less than 5.4 kg/m ² for women) according to the Asia Working Group for Sarcopenia (AWGS) standards. male SMI less than 7.0 kg/m ² ).

Bone mineral content (BMC) and bone mineral density (BMD) of the total femur, femoral neck, and lumbar spine (L1-L4) were measured using dual energy X-rays. Absorbance was measured by a trained technician using an absorptiometry (DXA, dual-energy X-ray absorptiometry, Lunar Prodigy, GE Healthcare, Madison, WI, USA). Osteoporosis was diagnosed using the T-score criteria of the World Health Organization (WHO) as supplied by the DXA manufacturer (Japan): normal, osteopenic, classified as osteoporosis. ① Normal if T-score ≥ -1 in any one of the three sites ② Osteopenia if -2.5 < lowest T-score < -1 ③ Osteopenia if lowest T-score ≤ -2.5 (osteoporosis).

Osteoporosis was defined as a BMD 2.5 standard deviation (SD) lower than the peak bone mass of a young, healthy, gender- and race-matched reference population according to the WHO diagnostic classification. The relationship between BMD (T-score) and SMI was used to classify osteopenia (T-score ≤ -2.5 and low SMI) and normal (high T-score and high SMI). Nutritional status was assessed preoperatively by a complete Mini nutritional assessment (MNA). The screening portion consists of questions about food intake, weight loss, mobility, stress, neuropsychological problems, and body mass index (BMI). did The evaluation part consisted of detailed questions about self-views on nutrition and health status, mid-arm and calf circumference measurements. A malnourished patient was defined as an MNA score of less than 17 points.

4. Experimental method

The library was prepared for 100 bp paired-end sequencing using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina, CA, USA). That is, mRNA molecules were purified and fragmented from 1 μg of total RNA using oligo(dT) magnetic beads. Fragmented mRNA was synthesized into single-stranded cDNA through random hexamer priming. This was applied as a template for second-strand synthesis to prepare double-stranded cDNA. After sequential processes of end repair, A-tailing, and adapter ligation, the cDNA library was amplified by PCR (Polymerase Chain Reaction). The quality of the cDNA library was assessed with an Agilent 2100 BioAnalyzer (Agilent, CA, USA). Libraries were quantified using the KAPA library quantification kit (Kapa Biosystems, MA, USA) according to the manufacturer's library quantification protocol. After cluster amplification of the denatured template, sequencing was performed with paired-end (2 × 100 bp) using the Illumina HiSeq platform sequencer (Illumina, CA, USA).

5. Transcriptome data analysis

Low quality reads were identified according to the following criteria; If more than 10% of the missing bases are included, if more than 40% of the bases with a quality score of less than 20 are included, if the average quality score of each read is less than 20. The entire filtering process was performed using an in-house script. Filtered short reads were mapped to the human reference coding sequence (CDS), version GRCh38 using the software Kallisto. Using Deseq2 to extract a set of differentially expressed genes (DEGs) that differentiate between sarcopenia and non-sarcopenia groups carefully defined by clinical observation through gene expression counts did to do Candidate genes were selected based on fold change and adjusted p-values.

6. Gene classification analysis

Public databases were used for classification of candidate genes in the DEG analysis; gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). GO annotations were downloaded from the Ensembl database; Human genes from Ensembl Genes 100 (GRCh38. p13). GO enrichment analysis was implemented by Fisher's exact test and the resulting p-value was corrected by fdr-bh of python module state models.

7. Quantitative real-time RT-PCR (qPCR)

As a template for PCR amplification, 1.3 μL of pre-synthesized cDNA was used for RNA sequencing. cDNA was prepared with forward and reverse primers for HPN of RUNX1, SLC25A3, BCL3, CHI3L1, MYBPH, ACSL5, NDUFB5, NGFR, PLAG2A, CSPG4, SEMA6B, TEP1, AC068506.1, THBS1, ACAT1, CYC1, TCAP in 384-well plates. (384-well plate: MicroAmp® 384-well Reaction Plate with Barcode with Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK), Applied Biosystems, Foster City, CA, USA). Each sample was measured 3 times. GAPDH was amplified by real-time RT-PCR (qPCR) as a housekeeping gene to normalize mRNA levels between samples. Analysis was performed using the ViiA™7 real-time PCR system (Applied Biosystems, USA). The sequences of the primers used in the experiment are shown in Table 1. The cycle threshold value measured after the experiment was analyzed by the relative quantification 2 ^-ΔΔC(T) method.

Gene Forward primer (5' → 3') sequence number Reverse primer (5' → 3') sequence number GAPDH AGCCACATCGCTCAGACAC One GCCCAATACGACCAAATCC 2 RUNX1 TCTTCACAAACCCACCGCAA 3 CTGCCGATGTCTTCGAGGTTC 4 SLC25A3 TATCTCTGGCGCACATCACTA 5 CTTAGCAGCTTCCATAGGAGC 6 BCL3 AACCTGCCTACACCCCTATAC 7 CACCACAGCAATATGGAGAGG 8 CHI3L1 GAAGACTCTCTTGTCTGTCGGA 9 AATGGCGGTACTGACTTGATG 10 MYBPH CCAGGGGAAGCCTAAGCCT 11 CGAGCGAATGAAGAGGATGGA 12 ACSL5 TGGCTATCTTACAAACAGGTGTC 13 TCCACTCTGGCCTATTCTGAG 14 NDUFB5 AGCTGGAAGTGCGAAAATTGA 15 ATACCAGGGTCCATCTCCTCT 16 NGFR CCTACGGCTACTACCAGGATG 17 CACACGGTGTTCTGCTTGT 18 PLA2G2A CTGTTGCTACAAACGTCTGGA 19 GCTCCCCGAGTTGCTAAACTT 20 CSPG4 GCCACGTTGTCAGTCGATG 21 CCCATAGGGGACCTCTAGGG 22 SEMA6B AAACGTGTGTCGGATGAAGGG 23 GGGTTGAAGGCGTTGGAAC 24 TEP1 TGGGTCGTTGGTTTGATTCAG 25 GCTTCACGGCCAGATCCTC 26 AC068506.1 CACGCACAGATGGAAGAAAT 27 CACCCAATCTTCCATTGCAT 28 THBS1 TGCTATCACAACGGAGTTCAGT 29 GCAGGACACCTTTTTGCAGATG 30 ACAT1 ATGCCAGTACACTGAATGATGG 31 GATGCAGCATATACAGGAGCAA 32 CYC1 CTTCGCGGGGTAGTGTTGG 33 GGCCAGACTTCGACGACAA 34 TCAP GTCGGAGGAGAACTGTGAGC 35 ATGCCCATCCGCATCATCAG 36 HPN GGGCCATTGTGGCTGTTCT 37 CGTCCCTTCCGTCTTGTCAAA 38

Experiment result

1. Comparison of parameters with and without osteosarcomenia

There was no significant difference between the two groups in age (P=0.638), grip strength (P=0.168), vitamin D (P=0.635), parathyroid hormone (P=0.754), and femoral neck BMD (P=0.626). Patient demographic data are shown in Table 2.

parameter Non-sarcopenia (n=5) sarcopenia (n=6) p-value Age (years) 79.89 ± 7.16 79.82 ± 9.30 0.638 BMI (kg/m2) 24.89 ± 7.16 21.83 ± 9.30 0.063 Grip strength (kg) 12.03 ± 5.00 9.58 ± 3.62 0.168 SMI (kg) 6.40 ± 0.65 4.83 ± 0.35 <0.001 Vitamin D (ng/ml) 11.30 ± 9.23 22.29 ± 9.10 0.635 PTH (ng/ml) 71.91 ± 38.92 84.24 ± 164.31 0.754 Smoking status 0 0 *Physical activity (moderate) One One 0.887 MNA score <17 3 4 0.819 BMI; body mass index, SMI; skeletal muscle mass, PTH; Parathyroid hormone, MNA; mini nutritional assessment, *Based on IPAQ scoring protocol: moderate-intensity activity 5 days a week

2. Genes related to diagnosis of osteosarcomenia

The collected samples were subjected to RNAseq using the Illumina platform. A total of 11 samples obtained from sarcopenia and non-sarcopenia groups were sequenced (Tables 3 to 5). Based on the number of reads generated and the mapping rate, we determined that the RNAseq results were reliable for downstream analysis (Table 6). The average amount of each sample read was 6.4 GB. More than 97% of the sequences were above the Q30 base quality score. Sequences were quantified by gene expression using Kallisto software. 15 genes (RUNX 1, NGFR, CH3L1, BCL3, PLA2G2A, MYBPH, TEP1, SEMA6B, CSPG4, ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP) were found to be statistically significant (adjusted p-value < 0.01) was identified as a differentially expressed gene (DEG) in both groups (Fig. 2A).

Sample ID TBI ID TotalReads TotalBases TotalBases
(Gb) GC_Rate KOB TN1808R1614 63532088 6416740888 6.42 GB 51.02% JOJ TN1808R1617 56211104 5677321504 5.68 GB 51.07% KOH TN1808R1610 62994258 6362420058 6.36 GB 51.10% LOJ TN1808R1611 68884364 6957320764 6.96 GB 51.06% KOS TN1808R1619 61560036 6217563636 6.22 GB 51.66% KOS TN1808R1633 66588492 6725437692 6.73 GB 50.99% AOJ TN1808R1620 67532796 6820812396 6.82 GB 51.44% JOJ1 TN1808R1622 57730692 5830799892 5.83 GB 51.71% SOJ TN1808R1632 69483628 7017846428 7.02 GB 51.95% LOO TN1808R1624 63021484 6365169884 6.37 GB 50.72% POJ TN1808R1630 61271252 6188396452 6.19 GB 51.78%

Sample ID N_ZeroReads N_ZeroReadsRate N5_LessReads N5_LessReadsRate N_Count N_Rate KOB 63281686 99.61% 63388090 99.77% 5234921 0.08% JOJ 55985420 99.60% 56079212 99.77% 4845202 0.09% KOH 62821580 99.73% 62903902 99.86% 3131012 0.05% LOJ 68622114 99.62% 68743304 99.80% 5280373 0.08% KOS 61325362 99.62% 61435496 99.80% 4554385 0.07% KOS 66340508 99.63% 66463756 99.81% 4528290 0.07% AOJ 67260840 99.60% 67371006 99.76% 5985049 0.09% JOJ1 57497104 99.60% 57587658 99.75% 5386047 0.09% SOJ 69223204 99.63% 69360150 99.82% 3942301 0.06% LOO 62794054 99.64% 62910706 99.82% 4066205 0.06% POJ 61041002 99.62% 61154054 99.81% 4299728 0.07%

Sample ID Q30_MoreBases Q30_MoreBasesRate Q20_MoreBases Q20_MoreBasesRate KOB 6249807627 97.40% 6330175310 98.65% JOJ 5533427287 97.47% 5603319517 98.70% KOH 6097382361 95.83% 6233291583 97.97% LOJ 6772778804 97.35% 6860910824 98.61% KOS 6048288752 97.28% 6128232037 98.56% KOS 6550166628 97.39% 6633723778 98.64% AOJ 6648049367 97.47% 6731779864 98.69% JOJ1 5677331808 97.37% 5752245208 98.65% SOJ 6812633558 97.08% 6905776448 98.40% LOO 6193412604 97.30% 6274874784 98.58% POJ 6037393953 97.56% 6109553856 98.73%

No Name Raw Clean Mapped Uniquely Mapped *Mapping Rate One KOH　(TN1808R1610) 62,994,258 62,282,548 60,243,478 58,662,846 0.942 2 LOJ　(TN1808R1611) 68,884,364 68,192,916 64,567,537 62,837,223 0.921 3 KOB　(TN1808R1614) 63,532,088 62,877,668 60,579,544 59,224,778 0.942 4 JOJ　(TN1808R1617) 56,211,104 55,670,042 52,612,367 50,931,286 0.915 5 KOS　(TN1808R1619) 61,560,036 60,895,764 56,119,137 54,433,258 0.894 6 AOJ　(TN1808R1620) 67,532,796 66,879,188 64,864,282 63,237,266 0.946 7 JOJA (TN1808R1622) 57,730,692 57,136,648 55,200,293 53,917,198 0.944 8 LOO　(TN1808R1624) 63,021,484 62,437,164 60,167,395 58,595,209 0.938 9 POJ　(TN1808R1630) 61,271,252 60,755,688 59,046,982 57,261,021 0.942 10 SOJ　(TN1808R1632) 69,483,628 68,607,470 59,649,061 57,876,042 0.844 11 KOS　(TN1808R1633) 66,588,492 65,989,738 62,839,413 61,223,425 0.928 * Number of clean reads / Number of uniquely mapped reads

Specifically, the expression patterns of DEGs were classified into three groups. Genes in group 1 showed increased expression in sarcopenia patients, especially in the first two cases, whereas pharopenia patients showed low expression levels. Genes in group 2 showed high expression in all sarcopenia patients compared to low expression in sarcopenia patients. On the other hand, the expression of genes in group 3 was increased in patients with sarcopenia. For functional annotation, DEGs were classified by the Kegg Brite database (Fig. 3A).

Sarcopenia and osteoporosis often present pathophysiological features such as infiltration of fat into muscle and bone. Commonly representative classes supporting this are “Exosome” and “Enzyme”, and genes commonly classified into both classes are long-chain acyl-CoA synthetase (ACSL) and acetyl-CoA C-acetyltransferase (ACAT). ACSL5 showed a clear upregulation, whereas ACAT1 showed a downregulation in sarcopenia patients (Fig. 2B). In particular, ACSL5 has a hub gene as a HuRI (Human Reference Interactome)-based interaction partner (Figure 4). The hub interactors of ACSL5 provide insight into the link between ACSL5 perturbations and sarcopenia. interacting hub genes; Solute carrier (SLC) families, transmembrane protein 14 B (TMEM14B) and aquaporin 6 (AQP6) imply transport-related protein activity may be associated with sarcopenia. In particular, another SLC family, SLC25A3, showed significant downregulation in patients with sarcopenia (Fig. 2B). Meanwhile, previous studies have shown changes in muscle and bone structure in response to high levels of fat and suggested increased levels of apoptosis and autophagy. Consistent with this result, HLA-F and HLA-DR3B showed differential expression and were mapped to the “Phagosome” (ko04145) pathway of the Kegg Pathway (Fig. 2B). Also DEG; HPN, RUNX1, MRPL45 and TCAP have the GO terms "macroautophagy" (GO:0016,236), "regulation of macroautophagy" (GO:0016,241) and "chaperone-mediated autophagy" (GO:0061,684) (Table S3 ), suggesting that patients with sarcopenia may have different autophagy regulation.

3. qPCR validation results

In the qPCR results, significant differences in BCL3, CHI3L1, MYBPH, ACSL5, NDUFB5, NGFR, PLA2G2A, CSPG4, SEMA6B, TEP1, AC068506.1, THBS1, ACAT1 and HPN gene expression levels were observed between the non-OS and OS groups. did not (FIG. 5). Although the expression levels of SLC25A3 and CYC1 genes in the OS group were significantly lower than those in the non-OS group, an increase in the RUNX1 mRNA level was observed in OS samples (p<0.05).

Claims (11)

RUNX 1 (RUNX family transcription factor 1), NGFR (nerve growth factor receptor), CH3L1 (Chitinase-3-like protein 1), BCL3 (B cell lymphoma 3), PLA2G2A (Phospholipase A2 Group IIA), MYBPH (myosin binding protein) H), TEP1 (telomerase associated protein 1), SEMA6B (semaphorin 6B), CSPG4 (chondroitin sulfate proteoglycan 4), ACSL5 (acyl-CoA synthetase long chain family member 5), SLC25A3 (solute carrier family 25 member 3), NDUFB5 ( Hip fracture comprising an agent measuring the expression level of at least one gene selected from the group consisting of NADH:ubiquinone oxidoreductase subunit B5), cytochrome c1 (CYC1), acetyl-CoA acetyltransferase 1 (ACAT1), and titin-cap (TCAP) (hip fracture) A composition for predicting whether a subject has sarcopenia. The composition according to claim 1, wherein the agent is at least one selected from the group consisting of primers, probes, antibodies, aptamers, DNA, RNA, proteins and polypeptides. The composition according to claim 1, wherein the hip fracture subject is accompanied by osteoporosis. A kit for predicting the onset of sarcopenia in a hip fracture subject comprising the composition of any one of claims 1 to 3. RUNX 1 (RUNX = family transcription factor = 1), NGFR (nerve growth factor receptor), CH3L1 (Chitinase-3-like protein 1), BCL3 (B cell lymphoma 3), PLA2G2A (Phospholipase A2) measured in samples isolated from the subject Group IIA), MYBPH (myosin binding protein H), TEP1 (telomerase associated protein 1), SEMA6B (semaphorin 6B), CSPG4 (chondroitin sulfate proteoglycan 4), ACSL5 (acyl-CoA synthetase long chain family member 5), SLC25A3 (solute carrier family 25 member 3), NDUFB5 (NADH: ubiquinone oxidoreductase subunit B5), CYC1 (cytochrome c1), ACAT1 (acetyl-CoA acetyltransferase 1) and TCAP (titin-cap) expression level of at least one gene selected from the group consisting of A method of providing information for predicting whether sarcopenia occurs in a hip fracture object, comprising the step of comparing the expression level in the control group. The method according to claim 5, wherein the expression level of at least one gene selected from the group consisting of RUNX 1, NGFR, CH3L1, BCL3 and PLA2G2A is higher than the expression level in the control group, compared to the control group. To provide information that the possibility of developing sarcopenia is high An information providing method, further comprising steps. The method according to claim 5, further comprising the step of providing information that if the expression level of at least one gene selected from the group consisting of MYBPH, TEP1, SEMA6B and CSPG4 is higher than the expression level in the control group, the possibility of developing sarcopenia will be higher compared to the control group. Including, how to provide information. The method according to claim 5, when the expression level of at least one gene selected from the group consisting of ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP is lower than the expression level in the control group, information that the possibility of developing sarcopenia compared to the control group is high A method for providing information, further comprising the step of providing. The method according to claim 5, wherein at least one selected from the group consisting of RUNX 1, NGFR, CH3L1, BCL3, PLA2G2A, MYBPH, TEP1, SEMA6B, CSPG4, ACSL5, SLC25A3, NDUFB5, CYC1, ACAT1 and TCAP in the sample isolated from the subject Further comprising the step of measuring the expression level of the gene, information providing method. The method according to claim 5, wherein the sample is one selected from the group consisting of muscle cells, muscle tissue, plasma, serum and urine. The method according to claim 5, wherein the hip fracture subject is accompanied by osteoporosis.

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