KR20230036504A - Markers for diagnosing Sarcopenia and use thereof - Google Patents

Abstract

The present invention relates to a method for providing information capable of diagnosing and predicting a risk group of sarcopenia in an early stage by providing an SNP marker capable of predicting a high risk of sarcopenia, a composition for diagnosing sarcopenia, and a kit including the same. The present invention includes an agent for detecting at least one single nucleotide polymorphism (SNP) selected from a group consisting of NCBI refSNP ID: rs1187118, rs3768582 and rs6772958.

Description

Markers for diagnosing sarcopenia and use thereof {Markers for diagnosing Sarcopenia and use thereof}

The present invention relates to a method for diagnosing sarcopenia by identifying a specific single nucleotide polymorphism (SNP) base having a significant correlation with sarcopenia, and a composition and kit for diagnosing sarcopenia, including an agent capable of identifying the SNP.

Sarcopenia is an age-related disorder in which muscle mass, strength, and function gradually decrease with age, and it is necessary to identify meaningful disease phenotypes and biomarkers related to sarcopenia when considering medical costs. .

Several studies have proposed various criteria for defining sarcopenia. Muscle mass is an important factor in the diagnosis of sarcopenia, and the heritability of muscle mass is estimated to be over 50%. Among parameters related to muscle mass, lean body mass (LBM) has been frequently used to predict sarcopenia. In addition, a recent study has shown that skeletal muscle index (SMI) and appendicular skeletal muscle mass (ASM) can also be reliable parameters (Non-Patent Document 1). In this respect, it is necessary to analyze SMI and ASM to understand the complex pathogenesis of sarcopenia, which can be attributed to various factors including oxidative stress, inflammation, mitochondrial dysregulation and genetic factors in addition to LBM (Non-Patent Document 2 and 3).

On the other hand, in humans, there is a mutation with a frequency of about once per 1000 bases, which is called a single nucleotide polymorphism (SNP), a 5% polymorphism is called a common polymorphism, and a 1-5% polymorphism is called a rare polymorphism. Currently, many experimental techniques have been developed to analyze the entire human nucleotide sequence, and among them, genome-wide association study (GWAS) has been used in many disease studies to date.

However, studies to diagnose the risk of sarcopenia using SNP polymorphisms associated with ASM or SMI have been rarely reported in the past, and most studies to investigate the genetic factors of LBM are not age-related and are based on European ancestry. it was done with

Chen LK, J Woo, P Assantachai, et al. (2020) Asian Working Group for Sarcopenia: 2019 Consensus Update on Sarcopenia Diagnosis and Treatment. J Am Med Dir Assoc 21: 300-307 e302. Roubenoff R (2003) Sarcopenia: effects on body composition and function. J Gerontol A Biol Sci Med Sci 58: 1012-1017. Rolland Y, S Czerwinski, G Abellan Van Kan, et al. (2008) Sarcopenia: its assessment, etiology, pathogenesis, consequences and future perspectives. J Nutr Health Aging 12: 433-450.

An object of the present invention is to provide a method for diagnosing sarcopenia or a method for providing information for diagnosis by identifying a specific SNP base having a significant correlation with the onset of sarcopenia with respect to a genetic sample obtained from a subject.

Another object of the present invention is to provide a composition or kit for diagnosing sarcopenia comprising an agent capable of identifying a specific SNP marker.

In order to achieve the above object, the present inventors hypothesize that individual SNP mutations associated with LBM, ASM, or SMI will affect the risk of developing sarcopenia, and compare sarcopenia patients with normal people to conduct a GWAS study , an association analysis was performed. As a result, the present invention was completed by discovering a single SNP biomarker capable of predicting and diagnosing the risk of sarcopenia in advance.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention provides a composition for diagnosing sarcopenia comprising an agent for detecting at least one single nucleotide polymorphism (SNP) selected from the group consisting of NCBI refSNP ID: rs1187118, rs3768582 and rs6772958.

The term "rs number" or "NCBI refSNP ID" of the present invention means a number indicating the sequence and position of the corresponding SNP for all SNPs for which NCBI is registered, and those skilled in the art can use the refSNP ID The chromosomal location where the SNP exists, the genetic loci, and the polymorphic sequence including the SNP can be easily identified. The specific sequence corresponding to the NCBI dbSNP number, refSNP ID, may be slightly changed according to the results of subsequent gene studies, and it will be apparent to those skilled in the art that the scope of the present invention also extends to the changed sequence.

Specifically, in the present invention, rs1187118 is a substitution of base 34,201,243 from A to G or T in the entire sequence of chromosome 6, and rs3768582 is base 183,584,735 of the entire sequence of chromosome 1, substitution from C to T, rs6772958 is a 32,007,599th base of the entire sequence of chromosome 3 substituted from A to G or T.

In the present invention, "polymorphism" refers to the case where two or more alleles exist in a single locus, and "polymorphic site" refers to the locus at which the alleles exist. Among the polymorphic sites, when only a single nucleotide differs from person to person, it is called a "single nucleotide polymorphism", that is, a single nucleotide polymorphism (SNP).

99.9% of genomic genes are common within individuals, and the remaining 0.1% is considered to be involved in individual differences related to the risk of developing a specific disease or hypersensitivity. . Since SNPs have a high frequency of occurrence and are distributed almost evenly throughout the genome, they are considered to be highly reliable genetic polymorphisms for studying the relationship between genes and the risk of onset or hypersensitivity. Therefore, when such changes in SNPs are effectively analyzed, information related to associations with various diseases related to natural genetic variation can be effectively analyzed, which can greatly contribute to screening of genetic diseases and personalized treatment.

The location of these SNPs is usually followed by highly conserved sequences. SNPs usually occur when one base at a specific position is replaced with another base, but it can also occur due to deletion or duplication of nucleotides. In particular, when the allele frequency is 5% or less, it is called a rare SNP, and when the allele frequency is 5% or more, it is called a common SNP. Rare SNPs may appear differently by ethnicity or race. The range of rare SNPs may change depending on whether the definition of these rare SNPs, a defined population, is viewed as the whole of mankind or limited to a specific group, and even if a variation is seen as a common SNP in one group, It is a self-evident fact that other groups can exhibit the appearance of rare SNPs.

As used herein, the term "allele" refers to several types of a gene present in the same locus of a homologous chromosome. Alleles are also used to indicate polymorphism, for example, SNPs have two types of alleles.

As used herein, the term "SNP marker" refers to a base pair of a single nucleotide polymorphism allele on a DNA sequence used to identify an individual or species. Since SNPs have a relatively high frequency, are stable, and are distributed throughout the genome, resulting in genetic diversity of individuals, SNP markers can serve as indicators of genetic proximity between individuals. SNP markers usually include phenotypic changes accompanying single nucleotide polymorphisms, but may not in some cases. In the case of the SNP markers of the present invention, variations in amino acid sequences or differences in phenotypes of individuals such as sarcopenia susceptibility may be indicated.

In the present invention, "subject" may mean a subject whose risk of sarcopenia is to be determined. Alternatively, the subject may have already developed sarcopenia. The sarcopenia can be diagnosed by analyzing the genotype of the SNP marker using a genetic sample obtained from the subject. The "sample" includes, but is not limited to, tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine obtained from a subject. A genetic sample may be obtained from the sample, and may include DNA, mRNA, or cDNA synthesized from mRNA.

As used herein, the term "diagnosis" refers to a series of actions for confirming the presence or characteristics of a disease in a subject. In the present invention, the diagnosis may include an act of confirming whether a disease has occurred. In addition, in the present invention, the diagnosis may include an act of confirming the risk of developing a disease. The present invention can be used to delay the onset or prevent the onset of sarcopenia through special and appropriate management for any specific subject at high risk of developing sarcopenia. In addition, the present invention can be used clinically to make treatment decisions by diagnosing sarcopenia at an early stage and selecting the most appropriate treatment modality.

In the present invention, the fact that the single nucleotide polymorphism marker can be used for diagnosing sarcopenia is based on the high probability that a specific base exists at the site of the single nucleotide polymorphism as a result of gene analysis of a group with sarcopenia. In an example of the present invention, the present inventors performed a GWAS meta-analysis on a total of 6,961 samples using multiple cohorts composed of elderly East Asians. Through this, it was confirmed that rs1187118, rs3768582, and rs6772958 were significantly related to the phenotype (LBM or ASM) associated with sarcopenia.

Specifically, in the present invention, rs1187118 or rs3768582 may be a genetic biomarker related to lean mass (LBM). In the present invention, rs6772958 may be a genetic marker associated with limb muscle mass (ASM).

The composition of the present invention may contain an agent for detecting or confirming one or more SNPs selected from the group consisting of rs1187118, rs3768582 and rs6772958 in a genetic sample isolated from a subject.

In the present invention, the agent is a polynucleotide consisting of 10 or more consecutive bases including the SNP or their complementary polynucleotide; Alternatively, it may be a probe or primer that specifically hybridizes with the polynucleotide.

For example, the agent contained in the composition of the present invention is preferably a polynucleotide consisting of 10 or more consecutive bases including one or more bases selected from the group consisting of rs1187118, rs3768582 and rs6772958, or a complementary polynucleotide thereof. can

In the present invention, "polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or unmodified DNA or modified RNA or modified DNA. Thus, for example, a polynucleotide as defined herein includes, but is not limited to, single and double-stranded DNA, DNA comprising single and double-stranded regions, single- and double-stranded RNA, and RNA comprising single- and double-stranded regions. , hybrid molecules comprising DNA and RNA that are single-stranded or more typically double-stranded, or may contain single- and double-stranded regions. Thus, DNA or RNA having a backbone that has been modified for stability or other reasons is a “polynucleotide” as the term is intended herein. Also, those containing unusual bases such as inosine or modified bases such as tritiated bases. DNA or RNA may be included in a “polynucleotide” as defined herein. In general, the term “polynucleotide” includes all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides. Polynucleotides can be produced by a variety of methods, including in vitro recombinant DNA-mediated techniques, and by DNA expression in cells and organisms.

The polynucleotide or its complementary polynucleotide according to the present invention may consist of 5 or more, preferably 10 to 100, more preferably 20 to 80, even more preferably 40 to 60 consecutive bases. However, it is not limited thereto.

As another example, the agent contained in the composition of the present invention may preferably be a polynucleotide that specifically hybridizes with the polynucleotide or its complementary polynucleotide.

In the present invention, the polynucleotide that specifically hybridizes with the polynucleotide or its complementary polynucleotide may be an allele-specific polynucleotide.

Specifically, an allele-specific polynucleotide means specifically hybridizing to each allele. That is, it refers to hybridization to be able to specifically distinguish the bases of the polymorphic site present in the polymorphic sequence. Here, hybridization conditions should be sufficiently stringent to hybridize only to specific alleles by showing significant differences in hybridization strength between alleles. Conditions suitable for hybridization may be determined by referring to information commonly known in the art, and may be determined by controlling temperature, ionic strength (buffer concentration), and the presence of a compound such as an organic solvent. These stringent conditions may be determined differently depending on the sequences to be hybridized.

In the present invention, the polynucleotide to specifically hybridize may be an allele-specific probe. "Probe" refers to a nucleic acid fragment capable of sequence-specific binding to a specific nucleic acid. A probe may be a nucleic acid fragment such as RNA or DNA corresponding to a few bases to several hundreds of bases as short as possible. (Labeling) to confirm the presence or absence of a specific nucleic acid Probes can be produced in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, etc. For the probe of the present invention, a sequence completely or partially complementary to a sequence containing a SNP may be used, but a sequence substantially complementary to a sequence that does not interfere with specific hybridization may be used. In the present invention, hybridization can be performed using a probe complementary to a site containing the SNP of the present invention, and the SNP can be identified through hybridization. Selection of an appropriate probe and hybridization conditions are known in the art base can be transformed.

In addition, in the present invention, the polynucleotide to specifically hybridize may be an allele-specific primer. In the present invention, a "primer" is a base sequence having a short free 3' terminal hydroxyl group, which can form base pairs with a complementary template, and refers to a short sequence that functions as a starting point for template strand copying, mainly amplifying a specific section. It is used in the form of a primer set that A primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. In addition, the primer may be composed of usually 7 to 50 or 15 to 30 bases, but the appropriate length of the primer may vary depending on the purpose of use. A primer may incorporate additional features that do not alter the basic properties of the primer that serve as the starting point of DNA synthesis. The primer sequence need not be perfectly complementary to the template, but must be sufficiently complementary to hybridize with the template. The primers can be used to amplify and detect a DNA fragment containing a polymorphic site by hybridizing to a sequence containing a SNP.

Primers or probes can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologs of a natural nucleotide, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoro amidates, carbamates, etc.) or to charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

The present invention provides a kit comprising an agent for detecting one or more SNPs selected from the group consisting of rs1187118, rs3768582 and rs6772958. The kit may be prepared by conventional methods known to those skilled in the art.

In one embodiment of the present invention, the kit may be an RT-PCR kit, a microarray chip, or a microfluidic chip kit. The kit of the present invention may include not only the polynucleotide or its complementary polynucleotide or the polynucleotide specifically hybridizing therewith, but also one or more other component compositions, solutions or devices suitable for the assay method.

As one embodiment, the kit of the present invention may be a kit including essential elements necessary to perform PCR, for example, each primer pair capable of amplifying a nucleic acid containing a SNP site, a test tube, or other Suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water and sterile water, etc. may additionally include,

As another embodiment, the kit of the present invention may be a kit for diagnosing sarcopenia that includes essential elements necessary for performing a DNA chip, preferably a microarray chip or a microfluidic chip kit. The DNA chip kit includes a polynucleotide specific to the SNP, or a substrate to which a primer or probe specifically hybridizing thereto is attached, and the substrate may include a nucleic acid corresponding to a quantitative control gene or a fragment thereof. In such a DNA chip kit, nucleic acids are regularly arranged on the surface of the chip, and multiple hybridization reactions occur between nucleic acids on the DNA chip and complementary nucleic acids contained in a solution treated on the chip surface, enabling mass parallel analysis.

The microarray may be composed of a conventional microarray except for including the polynucleotide of the present invention, its complementary polynucleotide, or a polynucleotide specifically hybridizing thereto. Hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. The detection is performed by, for example, labeling a nucleic acid sample with a fluorescent material, for example, a label material capable of generating a detectable signal including materials such as Cy3 and Cy5, followed by hybridization on a microarray and the labeling material. A hybridization result can be detected by detecting a signal arising from

A microfluidic chip is a microfluidic device that measures and analyzes the interaction of a material to be analyzed contained in a fluid sample with a biological material, cell, tissue, or detection device on the chip using microfluidic control technology. Processes such as hybridization, nucleic acid amplification, and capillary electrophoresis reactions can be miniaturized and compartmentalized to include

Sequencing, hybridization analysis by microarray, etc., amplification using PCR, etc. can be used to confirm the genotype of the SNP of the present invention.

Confirmation of the genotype of the SNP of the present invention can be performed by gene sequence analysis. For example, sequencing, mini-sequencing, automated sequencing, TaqMan analysis, pyrosequencing, allele specific PCR (allele specific PCR), dynamic allelespecific hybridization (DASH), PCR-RELP (restriction fragment length polymorphism), PCR-SSCP (single strand conformation polymorphism), PCR-SSO (specific sequence oligonucleotide), microarray hybridization, primer extension, Southern blot hybridization, dot hybridization, ASO combining PCR-SSO and dot hybridization Known methods such as (allele specific oligonucleotide) hybridization, rolling circle amplification (RCA), high resolution melting (HRM), or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) may be used. , but is not limited thereto.

Furthermore, the results of the SNP polymorphism can be statistically processed using a statistical analysis method commonly used in the art, for example, Student's t-test, Chi-Square test (Chi-Square test) square test), linear regression line analysis, multivariate logistic regression analysis, etc. for continuous variables, absolute variables, odds ratio, and 95 It can be analyzed using variables such as % confidence interval.

One aspect of the present invention provides an information providing method for diagnosing sarcopenia comprising the step of detecting at least one SNP selected from the group consisting of rs1187118, rs3768582 and rs6772958 in a genetic sample isolated from a subject. In addition, the present invention provides a method for diagnosing sarcopenia comprising detecting at least one SNP selected from the group consisting of rs1187118, rs3768582 and rs6772958 in a genetic sample isolated from a subject. In addition, the present invention provides a method for detecting one or more SNPs selected from the group consisting of rs1187118, rs3768582 and rs6772958 from a genetic sample isolated from a subject in order to provide information necessary for diagnosing sarcopenia.

All contents described in relation to the composition and kit for diagnosing sarcopenia above may be applied or applied mutatis mutandis to the above method.

The method may include comparing a SNP detected in a sample of a subject to a SNP control of a sarcopenia patient or a SNP control of a normal person. For example, if the SNP detected in the sample of the subject shows the same genotype as the SNP control of the sarcopenia patient, it can be determined that sarcopenia develops or has a high risk of developing sarcopenia, and if it does not show the same genotype as the control group, sarcopenia It may not occur or the risk of developing it may be judged to be low. Alternatively, if the SNP detected in the sample of the subject shows the same genotype compared to the SNP control group of a normal person, it may be determined that sarcopenia does not occur or the risk of developing sarcopenia is low, and if it does not show the same genotype as the control group, sarcopenia occurs or may be judged to be at high risk of developing the disease.

In the present invention, when one or more SNPs selected from the group consisting of rs1187118, rs3768582 and rs6772958 are detected, it may be diagnosed as having a high risk of sarcopenia. In one embodiment of the present invention, the method may include diagnosing that the risk of developing sarcopenia is higher when the base of rs1187118 is G or T than when the base is A. In another embodiment of the present invention, the method may include diagnosing that the risk of developing sarcopenia is higher when the base of rs3768582 is T than when the base is C. In another embodiment of the present invention, the method may include diagnosing that the risk of developing sarcopenia is higher when the base of rs6772958 is G or T than when the base is A.

Advantages and features of the present invention, and how to achieve them, will become clear with reference to the detailed description of the following embodiments. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various different forms, and only the present embodiments will complete the disclosure of the present invention and allow common knowledge in the art to which the present invention belongs. It is provided to fully inform the holder of the scope of the invention, and the present invention is only defined by the scope of the claims.

The present invention relates to a method, composition, and kit for providing information capable of diagnosing sarcopenia. SNP mutations associated with the risk of developing sarcopenia were identified through genome-wide genome association analysis (GWAS). It can be used for diagnosis or disease prediction.

Figure 1 shows a schematic diagram of the assay study design.
Figure 2 shows a Manhattan plot for LBM.
3 shows a QQ plot for LBM.
Figure 4 shows a Manhattan plot for ASM.
5 shows a QQ plot for ASM.
6 is a LocusZoom plot of rs1187118, a genome-wide significantly associated SNP for LBM.
7 is a LocusZoom plot of rs3768582, a genome-wide significantly associated SNP for LBM.
8 is a LocusZoom plot of rs6772958, a genome-wide significantly associated SNP for ASM.

Hereinafter, the present application will be described in detail through examples. The following examples are merely illustrative of the present application, but the scope of the present application is not limited to the following examples.

[Example]

This study aimed to investigate genetic variations associated with sarcopenia in an elderly cohort using a genome-wide association study (GWAS) meta-analysis of both lean body mass (LBM) and body fat mass in 6,961 subjects. did. To this end, we analyzed two genetic cohorts of elderly Koreans, and subgroup GWAS was also performed for appendicular skeletal muscle mass (ASM) and skeletal muscle index (SMI).

The impact of significant single nucleotide polymorphisms (SNPs) on gene expression was also investigated using differential expression genetic analysis of the multiple expression quantitative trait loci (eQTL) data set, Gene Expression Omnibus data set (GSE38718).

Experiment method

1-1. subject of study

Data were obtained from two cohorts: VHSMC (n=2,518) and KARE (Ansan/Anseong Study: Korean Genome and Epidemiology Cohort, n=5,235). The VHSMC cohort is a hospital-based elderly cohort that includes many subjects with various diseases, and the KARE cohort is a nationally representative cohort for genome research in Korea, which is a longitudinal cohort of Ansan and Anseong communities in Korea. This study included subjects from the KARE cohort and the VHSMC cohort with data on the use of a Korean chip (Korea Biobank Array Affymetrix Axiom 1.1 (Affymetrix, Santa Clara, CA)) developed by the National Institute of Health Genome Center. Subjects with reduced or limited function or chronic diseases that could affect primary sarcopenia according to AWGS 2019 were excluded, and 6,961 participants were enrolled across both cohorts (FIG. 1). The Institutional Review Board (IRB) of VHSMC approved the study protocols (IRB No.2020-02-015 and IRB No.2021-05-005), and the study was conducted in accordance with the Declaration of Helsinki. Both the VHS Biobank Committee (VBP-2020-03) and the National Biobank of Korea (KBN-2021-041) approved the use of biomass for this study.

1-2. Muscle Mass Measurement Assessment

Bioelectrical impedance analysis (BIA) measurements were performed using the InBody 770 (Biospace Co., LTD, Seoul, Korea) in the VHSMC cohort and the InBody 3.0 (Biospace Co., LTD, Seoul, Korea) in the KARE cohort. The screen automatically displays measurements of LBM (kg), skeletal muscle mass (kg), body fat mass (BFM; kg), and body fat percentage (%). LBM and BFM data were available for both cohorts and were used as initial phenotypes for analysis. Subgroup analyzes were performed using ASM or SMI derived from BIA; These data were only available for the VHSMC cohort. Parameters were defined according to the consensus of AWGS 2019.

1-3. Genotype and substitution

Genomic DNA was isolated from venous blood samples, and 100 ng of DNA genotype was analyzed using a Korean chip (Korea Biobank Array Affymetrix Axiom 1.1 (Affymetrix, Santa Clara, CA)) designed by the National Institute of Health. Genotypes were identified with a K-medoid clustering-based algorithm to minimize batch effects. The PLINK (version 1.9, Boston, MA) and ONETOOL (Song et al. 2018, Ref. 1) software packages were used for quality control procedures and association analyses. Samples meeting one of the following criteria were excluded: (1) gender mismatch, or (2) a call rate of up to 97%. SNPs were filtered out if their call rates were lower than the Hardy-Weinberg equilibrium (HWE) permutation test (P < 1Х10 ^-5 ).

Genotype replacement was performed using the Michigan imputation server. Only the 'non-European' or 'mixed' populations of the Haplotype Reference Consortium (Haplotype Reference Consortium release v1.1, McCarthy et al. 2016, Ref. 2) were used for reference purposes, respectively Eagle v2.4 (Loh et al. 2016, Ref. 3) and Minimac4 (Das et al. 2016, Ref. 4) were used to perform pre-phasing and imputation. After the imputation process, the R-squared (Rsq) is less than 0.9 or there are overlapping SNPs, the missing genotype rate is greater than 0.05, the P-value for HWE is less than 1Х10 ^-5 , or SNPs that were substituted were removed if their minority allele frequency (MAF) was less than 0.05. MAF was compared with reference data such as the Korean Reference Data (Kref) or the Genome Aggregation Database (GnomAD) of East Asian subjects. Finally, 2,422 subjects (and 2,804,834 SNPs) from the VHSMC cohort and 5,235 subjects (and 3,423,819 SNPs) from the KARE cohort were used for analysis.

1-4. statistical analysis

Baseline characteristics of the study population are presented here as means with standard deviations (SDs) for continuous variables and numbers, and as ratios for categorical variables. Genome analysis was performed using a linear model, and PLINK was used within each cohort. Age, sex and 10 principal component scores were included as covariates.

A meta-analysis of the VHSMC and KARE cohorts was performed using METAL software. Cochran's Q-test for heterogeneity was performed; P-values were expressed as 'HetPVal''. Genes near significant SNPs and loci of each GWAS were plotted using the software. The threshold for statistical significance in this model was P < 5.0Х10 ^-8 , which is generally considered to reflect the significance of the whole genome.

1-5. Comparative genomic analysis (Functional annotation)

Expression quantitative trait loci (eQTL) studies were performed using the genotype-tissue expression (GTEx) dataset, which provides diverse human tissues from donors of identically clustered genotypes to assess genetic variation within their genomes. In the gene expression omnibus (GEO) dataset (GSE38718), we further investigated the relevant genes as differentially expressed genes (DEGs) in the skeletal muscle of subjects aged 19-28 and 65-76 years. A significance level with a false discovery rate (FDR) adjusted to 0.1 was applied using the Benjamini-Hochberg correction to correct for multiple hypothesis testing.

Experiment result

1. Characteristics of study participants

The characteristics of study participants are shown in Table 1. In the table below, a written as a superscript means a Chi-square test, and b means a t-test.

2. GWAS meta-analysis of lean mass (LBM)

A total of 2,360,975 SNPs were used in the GWAS meta-analysis of LBM. Manhattan plots and quantile-quantile (Q-Q) plots for LBM are shown in FIGS. 2 and 3 , respectively. The Q-Q plot revealed no evidence for inflation of the test statistic (variance inflation factor [VIF] = 1.044). The top 10 variants of LBM are listed in Table 2. In the table below, superscripted a indicates Korean reference data (Kref), and b indicates Genome Aggregation Database (GnomAD) (East Asians).

Two of these were significant loci across the genome. The most significant mutation was rs1187118 near Glutamate Metabotropic Receptor 4 (GRM4) and High Mobility Group AT-Hook 1 (HMGA1) (effect = 0.720, standard error [SE] = 0.117, P = 1.09 × 10-9, HetPVal = 0.199 ), followed by rs3768582 near NCF2 (Neutrophil Cytosolic Factor 2) (effect = 0.554, SE = 0.100, P = 4.09 × 10 -8, HetPVal = 0.537).

3. GWAS of limb skeletal muscle (ASM)

A total of 2,804,834 SNPs were used in the GWAS analysis of ASM using only the VHSMC cohort. Manhattan and Q-Q plots for ASM are shown in Figures 4 and 5, respectively; A Q-Q plot revealed no evidence for inflation of the test statistic (VIF=1.031). The top 10 variants of ASM are listed in Table 3. In the table below, a in superscript is the Veterans Health Service Medical Center (VHSMC), b is the Korean reference data (Kref), and c is the Genome Aggregation Database (GnomAD) ( East Asian). Also, MAF means minor allele frequency.

The only significant mutation was rs6772958 (effect = -0.456, SE = 0.081, P = 2.30 × 10-8), a full-length genomic locus near ZNF86 (zinc finger protein 86) and GPD1L (Glycerol-3-Phosphate Dehydrogenase 1 Like). was

4. Positional Analysis and Functional Annotation

Regional plots with major SNPs for significant genome-wide variation of each phenotype (LBM and ASM) are shown in Figures 6-8.

Regarding LBM, rs1187118 eQTL analysis of the GTEx project (V7), which is the most significant SNP across the genome, showed that RPS10 (Ribosomal Protein S10) was highly expressed in sunlight-exposed lower extremity tissues (P = 1.40 × 10 ^-7 ). In addition, linkage disequilibrium (LD) variant eQTL association to Nudix Hydrolase 3 (NUDT3) was also found in skeletal muscle tissue (P = 4.30×10 ^-21 ). RPS10 is a gene upstream of NUDT3 and may have a regulatory function for the association between NUDT3 and sarcopenia. In the present invention, NUDT3 was found to be associated with LBM in an elderly cohort. NUDT3 belongs to the MutT or Nudix protein family, which serves as a homeostatic checkpoint at critical steps in the inositol phosphate metabolic pathway. Thus, pathways such as phosphatidyl-1D-myo-inositol and glycerophospholipid metabolism from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database may be related to LBM.

Second, analysis of rs3768582 eQTL, a significant SNP throughout the genome, showed that NCF2 (Neutrophil Cytosolic Factor), SMG7 (SMG7 Nonsense Mediated mRNA Decay Factor), and ARPC5 (Actin Related Protein 2/3 Complex Subunit 5) were significantly 10 ⁻⁹ ), heart (P = 2.60×10 ⁻⁵ ) and cell tissue (P = 7.30×10 ⁻⁵ ), respectively. A study on DEGs in skeletal muscle tissue from patients with cachexia (Narasimhan et al. 2018, Ref. 5) showed that NCF2 was identified in signaling pathways related to inflammation, which is consistent with the results of this study. SMG7 encodes a protein essential for nonsense-mediated mRNA decay known to be associated with height and BMI-adjusted waist circumference in the GWAS catalog. Since SMG7 is linked to telomerase reverse transcriptase (TERT), sarcopenia may be related to muscle cell senescence through microRNA-195. In addition, ARPC5 encodes the actin-related protein 2/3 complex, which shows a negative fold difference in expression associated with the cytoskeleton of muscle tissue.

Meanwhile, in the DEG analysis using GSE38718, RPS10 (P = 8.00×10 ^-4 ), NUDT3 (P = 1.19×10 ^-3 ), NCF2 (P = 1.26×10 ^-2 ), SMG7 (P = 1.03×10 ^-3 ) and ARPC5 (P = 4.26×10 ^-2 ) were more expressed in the elderly group (Table 4) than in the young group.

Regarding ASM, analysis of rs6772958 eQTL, the only significant SNP in the whole genome, showed that GPD1L was highly expressed in thyroid tissue (P = 8.10×10 ^-15 ). A tissue-based study of rat muscle identified GPD1L as a candidate locus for sarcopenia. These results were also observed in a previous study examining sarcopenic muscle tissue in elderly women, where GPD1L was found to be downregulated through cellular energy metabolism. In addition, a systemic genetic approach confirmed that the molecular mechanisms of GPD1L and human adipose tissue obesity are related to energy metabolism. GPD1L expression was found to negatively correlate with microRNA-210 (miR-210) levels and was consistently downregulated in obese subjects. They hypothesized that reduced miR-210 levels would increase GPD1L and suppress hypoxic transcription factor-1α (HIF-1α) activity. A previous study of miRNAs circulating in plasma showed that miR-210 was significantly downregulated in elderly patients with sarcopenia compared to patients without sarcopenia (He et al. 2020, Ref. 6). In summary, GPD1L can be a genetic biomarker for sarcopenia based on the miR-210 and HIF-1α pathways.

[references]

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4: Das S, L Forer, S Schonherr, et al. (2016) Next-generation genotype imputation service and methods. Nature Genetics 48: 1284-1287.

5: Narasimhan A, R Greiner, OF Bathe, et al. (2018) Differentially expressed alternatively spliced genes in skeletal muscle from cancer patients with cachexia. J Cachexia Sarcopenia Muscle 9: 60-70.

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The present invention has been described with reference to the above-described embodiments and experimental examples, but this is only exemplary, and those skilled in the art can make various modifications and equivalent other examples and experimental examples therefrom. will understand Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

Claims (6)

NCBI refSNP ID: comprising an agent for detecting one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of rs1187118, rs3768582 and rs6772958,
A composition for diagnosing sarcopenia. According to claim 1,
The agent is a probe or primer capable of detecting one or more SNPs selected from the group consisting of rs1187118, rs3768582 and rs6772958,
Composition for diagnosis of sarcopenia Comprising the composition of claim 1,
A kit for diagnosing sarcopenia. According to claim 3,
The kit is an RT-PCR kit, a microarray chip or a microfluidic chip,
A kit for diagnosing sarcopenia. For a genetic sample isolated from a subject,
Detecting one or more SNPs selected from the group consisting of rs1187118, rs3768582 and rs6772958 using the composition of claim 1,
Methods of providing information for the diagnosis of sarcopenia. According to claim 5,
When one or more SNPs selected from the group consisting of rs1187118, rs3768582 and rs6772958 are detected, it is diagnosed as having a high risk of developing sarcopenia,
Methods of providing information for the diagnosis of sarcopenia.

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