KR20230138804A - A novel Charcot-Marie-Tooth disease diagnostic biomarker comprising a mutant of the glycyl-tRNA Synthetase 1 gene and Uses Thereof - Google Patents

Abstract

The present invention relates to a c.2171C>A mutant of glycyl-tRNA synthetase 1 (GARS1) gene and a method for diagnosing Charcot-Marie-Tooth disease (CMT) using the same. Specifically, it was confirmed of a novel c.2171C>A mutant of the GARS1 gene and it was identified that the mutant is the causative gene of the CMT. The GARS1 gene mutant according to the present invention may be effectively used for diagnosing CMT.

Description

Novel Charcot-Marie-Tooth disease diagnostic biomarker comprising a mutation of the glycyl-tRNA synthetase 1 gene and use thereof {A novel Charcot-Marie-Tooth disease diagnostic biomarker comprising a mutant of the glycyl-tRNA Synthetase 1 gene and Uses Thereof}

The present invention provides a novel Glycyl-tRNA Synthetase 1 ( GARS1 ) gene causal mutation biomarker for diagnosing Charcot-Marie-Tooth disease, a diagnostic composition using the same, a diagnostic kit, or a Charcot-Marie-Tooth disease using the same. This is about the method of diagnosing Tooth disease.

Charcot-Marie-Tooth disease (CMT), or hereditary motor and sensory neuropathy, refers to a disease in which motor and sensory nerves are damaged by specific gene mutations. Inherited peripheral neuropathies are classified into three categories: Hereditary motor and sensory neuropathies (HMSN), Hereditary motor neuropathies (HMN), and Hereditary sensory neuropathies (HSN). Hereditary motor and sensory neuropathies are It is one of two things. After it was first reported by Charcot, Marie and Tooth in 1886, it was named Charcot-Marie-Tooth disease, or CMT for short. Dyck in the second half of the 20th century. et al. changed the name from CMT to hereditary motor and sensory neuropathy, and currently, CMT and HMSN are used together.

The incidence of CMT is 1 in 2,500 people, which is the highest frequency among inherited rare diseases. In CMT patients, the muscles in the feet and hands gradually atrophy, resulting in weakened strength, deformed foot and hand shapes, facial paralysis, and hearing impairment. Onset usually occurs around the age of 10, but rarely occurs after the age of 30. Depending on the type of genetic mutation, patients' symptoms range from mild, almost normal, to very severe, requiring assistance with walking or relying on a wheelchair.

Depending on the pattern of inheritance, CMT is divided into myelin defect type (CMT1), axonal type (CMT2), intermediate type (IntCMT) with autosomal dominant and recessive inheritance, and CMTX type with X-chromosome-linked inheritance. In the case of CMT type 1, it was named 1A, 1B, 1C, etc. in the order in which genetic mutations were reported. To date, more than 100 causative genes have been reported as the cause of CMT. It is caused by a single gene mutation in the causative gene, and although the severity of symptoms varies, it is inherited according to Mendel's laws. In addition, since genetic defects and onset are directly related, the results of genetic diagnosis of the cause of CMT have the meaning of confirmation rather than a simple tendency or possibility of onset, making it very appropriate to utilize a genetic diagnosis system.

Existing CMT treatments were mainly limited to rehabilitation treatment, assistive devices, and pain control, but the discovery of related genes made genetic counseling and family planning possible, and clinical treatment attempts based on scientific evidence are gradually developing. . Currently, there is still a lack of practical treatment or support that can change the progression of inherited motor-sensory neuropathy, but recent animal experiments have shown promising results.

Among the drugs for CMT treatment, there was a report that administration of onapristone, a progesterone receptor antagonist, reduced overexpression of Pmp22 mRNA in transgenic mice and improved the phenotype of hereditary motor-sensory neuropathy without side effects, and in peripheral nerves. Ascorbic acid, an essential substance for myelin formation, improved myelin remodeling and the phenotype of hereditary motor-sensory neuropathy in CMT1A transgenic mice, and neurotrophin-3 (NT-3) improved the phenotype in CMT1A patient groups. It was reported that it improved sensory symptoms by increasing myelinated nerve fibers. Therefore, accurate genetic diagnosis of CMT has made it possible to suppress its onset and provide customized treatment appropriate for the genetic cause, but the development of efficient diagnostic methods for hereditary neurological diseases is still insufficient. In order to provide customized treatment for motor-sensory neuropathy, which has genetically very heterogeneous characteristics, an accurate genetic diagnosis method is first required to be established.

While researching the causative gene of Charcot-Marie-Tooth disease, the present inventors identified a mutation in a specific gene, confirmed that the mutation was the causative gene of Charcot-Marie-Tooth disease, and completed the present invention.

Therefore, the purpose of the present invention is to provide a biomarker composition for diagnosing Charcot-Marie-Tooth disease (CMT).

Another object of the present invention is to provide a composition for diagnosing Charcot-Marie-Tooth disease.

Another object of the present invention is to provide a kit for diagnosing Charcot-Marie-Tooth disease.

Another object of the present invention is to provide a method of providing information for diagnosing or predicting the risk of developing Charcot-Marie-Tooth disease.

Another object of the present invention is to provide a screening method for a treatment for Charcot-Marie-Tooth disease.

In order to achieve the above object, the present invention treats Charcot-Marie-Tooth disease, which includes genetic causative mutations in the Glycyl-tRNA Synthetase 1 ( GARS1 ) gene. CMT) provides a diagnostic biomarker composition.

In order to achieve the above object, the present invention provides a treatment for Charcot -Marie-Tooth disease ( Charcot- A biomarker composition for diagnosing Marie-Tooth disease (CMT) is provided.

In addition, in order to achieve the above other objects, the present invention provides an agent capable of detecting a GARS1 mutant gene containing the 2,171st mutation site in the base sequence shown in SEQ ID NO: 1 or a GARS1 mutant protein encoded by the GARS1 mutant gene. Provided is a composition for diagnosing Charcot-Marie-Tooth disease (CMT), including.

In addition, in order to achieve the above other object, the present invention provides a diagnostic kit for Charcot-Marie-Tooth disease containing the diagnostic composition according to the present invention.

In addition, in order to achieve the above further object, the present invention provides a method for treating Charcot-Marie-Tooth disease, comprising the step of detecting a GARS1 mutant gene or a GARS1 mutant protein encoded by the GARS1 mutant gene in a biological sample isolated from a subject. Provides a method of providing information for diagnosing or predicting the risk of developing (Charcot-Marie-Tooth disease).

In addition, in order to achieve the above other object, the present invention provides a screening method for a treatment for Charcot-Marie-Tooth disease comprising the following steps: (a) treating target cells with a candidate for the treatment of Charcot-Marie-Tooth disease; step; (b) measuring whether a GARS1 mutant gene or a GARS1 mutant protein encoded by the GARS1 mutant gene is detected in the cells of step (a); and (c) determining it as a treatment for Charcot-Marie-Tooth disease when the GARS1 mutant gene or GARS1 mutant protein is not detected in the cells measured in step (b).

The c.2171C>A mutation in the Glycyl-tRNA Synthetase 1 ( GARS1 ) gene according to the present invention is a novel biomarker for Charcot-Marie-Tooth disease (CMT). As such, diagnosis using the GARS1 mutant gene or the protein encoded by the GARS1 mutant gene enables accurate early diagnosis of Charcot-Marie-Tooth disease through genetic testing, and accordingly, treatment methods that have recently been developed can be applied at an early stage. The treatment effect can be maximized by applying it.

Figure 1 shows the pedigree and genotype of a CMT patient family with an ARS gene mutation identified in the present invention. A. is the result of a CMT patient family with a GARS1 gene mutation, and B. is the result of HARS1 (Histidyl-TRNA Synthetase 1). C. is the result of a family of CMT patients with a gene mutation, C. is the result of a family of CMT patients with a WARS1 (Tryptophanyl-TRNA Synthetase 1) gene mutation, and D. is a result of a CMT patient with a YARS1 (Tyrosyl-TRNA Synthetase 1) gene mutation. It is a result of family. In Figure 1, □ and ○ represent unaffected members, ■ and ● represent affected members, and ^ represents twins. Gray circles represent cases with pathogenic mutations but very mild symptoms, and arrows represent probands.
Figure 2 shows the results of sequencing and conservation analysis of ARS gene mutations. The causative mutation identified by whole exome sequencing (WES) was confirmed through Sanger sequencing. Vertical arrows indicate mutation sites, WT indicates the wild-type allele, and Mut indicates the mutant allele.
Figure 3 shows the schematic structure of the ARS protein, where A. is the result of the GARS1 protein, B. is the result of the HARS1 protein, C. is the result of the WARS1 protein, and D. is the result of the YARS1 protein. This is the result (DHHA1 (DHH subfamily 1 member), RNA binding function, MTS (mitochondria targeting sequence), SAD (tRNA synthetase second additional domain (tRNA synthetase second additional domain), TRBD (tRNA binding domain), WHEP (domain showing high affinity interaction with tRNA)). Pathogenic mutations identified in the present invention are indicated in red at the bottom of the diagram, and some reported mutations are indicated in black at the bottom of the diagram.
Figure 4 shows the results of predicting 3D conformational changes surrounding pathogenic mutations in the ARS protein, for p.P336H and p.P724H of GARS1 . Wild-type ARS protein residues (top) and mutant amino acids (bottom) are shown in pink, hydrogen bonds are shown as dotted lines, and carbon, hydrogen, nitrogen, and oxygen are shown in green, white, blue, and red, respectively.
Figure 5 shows the results of comparing clinical phenotypes between patients classified by ARS gene ( GARS1 and YARS1 ) mutations, where A. is age of onset (AOO), B. is FDS, C. is CMTNS, and D. is The median MNCV, E. and F., are the results of muscle weakness in the arms and legs, and G. and H. are the results of jerks in the knees and ankles.
Figure 6 shows the results of analyzing the correlation of phenotypic distribution of patients according to age of onset, where A is AOO versus functional disblility scale (FDS), B is AOO versus CMT neuropathy score (CMTNS), and C. is AOO versus median motor nerve conduction velocities (MNCVs), D. is AOO versus median compound muscle action potentials (CMAP), and E is AOO versus median sensory nerve conduction velocities. nerve conduction velocities (SNCV), F is the result of AOO versus median sensory nerve action potential (SNAP).

Hereinafter, the present invention will be described in detail.

The present invention provides a biomarker composition for diagnosing Charcot-Marie-Tooth disease (CMT), which includes a genetic causal mutation in the Glycyl-tRNA Synthetase 1 ( GARS1 ) gene. to provide.

In the present invention, “mutation” refers to a phenomenon that causes a qualitative or quantitative change in a gene due to an abnormality in a gene or chromosome, causing a change in genetic characteristics. The mutation may preferably be a gene-level mutation, and more preferably may be a point mutation or multiple mutation selected from substitution, insertion, and deletion. Additionally, the mutation may be a silent mutation, a neutral mutation, a missense mutation, a nonsense mutation, a frameshift mutation, etc., and the types thereof are not limited.

In the present invention, the “Glycyl-tRNA Synthetase 1 ( GARS1 ) gene” is one of aminoacyl-tRNA synthetase, and is used in aminoacyl-tRNA and related compounds. It encodes a glycyl-tRNA synthetase that forms a carbon-oxygen bond, and may preferably include the base sequence represented by SEQ ID NO: 1, and may be a partial sequence described in NCBI Accession number: NM_002047.4. More preferably, the base sequence represented by SEQ ID NO: 1 may have A (45th) of ATG, the start codon, as the first base among the base sequences listed in NCBI Accession number: NM_002047.4.

According to one embodiment of the present invention, more specifically, a novel c.2171C>A mutation was identified in the GARS1 gene represented by SEQ ID NO: 1, that is, a mutation in which the 2,171st base was substituted from C (cytosine) to A (adenine). It has been done. In addition, it has been confirmed that the above mutation is the causative gene of Charcot-Marie-Tooth disease, so it can be usefully used as a marker for diagnosing Charcot-Marie-Tooth disease.

In the present invention, “diagnosis” includes confirming the presence or absence of a disease, the state of the disease, and the prognosis of the disease, and refers to all types of analysis used to derive the disease state and decision. The novel genetic causative mutation of the GARS1 gene identified in the present invention is a genetic mutation that is closely related to the onset of Charcot-Marie-Tooth disease, and confirming the mutation of the GARS1 gene is useful in diagnosing Charcot-Marie-Tooth disease. can be used

The selection and application of meaningful diagnostic markers can determine the reliability of diagnostic results. A meaningful diagnostic marker may mean a marker that has high validity because the results obtained from diagnosis are accurate and has high reliability so that it shows consistent results even when measured repeatedly. As the diagnostic marker, the c.2171C>A mutation of the GARS1 gene has been confirmed as the causative mutation in patients with Charcot-Marie-Tooth disease, and diagnosis is made based on the results obtained by detecting the presence of the c.2171C>A mutation of the GARS1 gene. The results obtained are reasonably reliable.

In addition, the present invention relates to Charcot-Marie-Tooth disease (CMT), which includes a GARS1 mutant protein encoded by a GARS1 mutant gene containing the 2,171st mutation site in the nucleotide sequence shown in SEQ ID NO: 1. ) Provides a diagnostic biomarker composition.

The GARS1 mutant protein is a mutant protein (p.P724H) encoded by a gene (c.2171C>A) in which the 2,171st base of the GARS1 gene, shown in SEQ ID NO: 1, is substituted from C (cytosine) to A (adenine). ) is characterized in that. In other words, the mutant protein may mean a protein in which the 724th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is substituted from P (proline) to H (histidine).

The amino acid sequence represented by SEQ ID NO: 2 may be NCBI Accession number: NP_002038.2.

In addition, the present invention provides a Charcot-Marie- A composition for diagnosing Charcot-Marie-Tooth disease (CMT) is provided.

More preferably, when the 2,171st mutation site in the base sequence of the GARS1 gene is adenine, Charcot-Marie-Tooth disease is diagnosed. Alternatively, if the 724th amino acid in the amino acid sequence of the GARS1 protein is H (histidine), Charcot-Marie-Tooth disease is diagnosed.

The agent consists of a primer, a probe, and an anti-sense nucleotide that specifically binds to the GARS1 mutant gene mRNA, including the 2,171st mutation site in the base sequence shown in SEQ ID NO: 1. It may be one or more types selected from the group, but is not limited thereto.

In the present invention, the term "primer" refers to a nucleic acid sequence with a short free 3'-hydroxyl group that can form a base pair with a complementary template and copies the template strand. refers to a short nucleic acid sequence that serves as a starting point for Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer and temperature. In the present invention, CMT can be diagnosed or predicted by determining whether the desired product is produced by performing PCR amplification using sense and antisense primers of GARS1 polynucleotide. PCR conditions and lengths of sense and antisense primers can be appropriately modified based on those known in the art.

In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA that can bind specifically to mRNA, ranging from a few bases to several hundred bases in length, and is labeled. Therefore, the presence or absence of a specific mRNA can be confirmed. Probes may be manufactured in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, etc.

Primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologs, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphosotriester, phosphatase). phoramidate, carbamate, etc.) or charged linkages (e.g. phosphorothioate, phosphorodithioate, etc.).

The primer or probe preferably contains 8 or more nucleotides, and the hybridization method can be achieved by exposing or contacting the primer or probe to the 2,171st mutation site in the base sequence of the GARS1 gene of the present invention. . Preferably, these sequences are hybridized under conditions appropriately controlled to minimize non-specific binding; for example, suitable conditions for detection of about 80% to 90% identical sequences are 0.25M Na ₂ HPO ₄ , pH 7.2, 6.5. % SDS, overnight hybridization at 42°C in 10% dextran sulfate, and a final wash at 55°C in 0.1 Υ SSC, 0.1% SDS. Additionally, suitable conditions for detection of about 90% or more identical sequences are hybridization overnight at 65°C in 0.25M Na ₂ HPO ₄ , pH 7.2, 6.5% SDS, 10% dextran sulfate, and 0.1 Υ SSC in 0.1% SDS. , which may include a final wash at 60°C.

Alternatively, the agent is an oligopeptide, monoclonal antibody, polyclonal antibody, or chimeric that specifically binds to the protein encoded by the GARS1 mutant gene containing the 2,171st mutation site in the base sequence shown in SEQ ID NO: 1. It may be one or more types selected from the group consisting of (chimeric) antibodies, ligands, PNA (peptide nucleic acid), and aptamers, but is not limited thereto.

In the present invention, the term “antibody” is a term known in the art and refers to a specific protein molecule directed to an antigenic site. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to a protein encoded by the GARS1 mutant gene, which is a marker of the present invention, and such antibody is produced by cloning each gene into an expression vector according to a conventional method. The protein encoded by the marker gene can be obtained and produced from the obtained protein by a conventional method. This also includes partial peptides that can be made from the above proteins, and the partial peptides of the present invention include at least 7 amino acids, preferably 9 amino acids, and more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited, and as long as it is a polyclonal antibody, monoclonal antibody, or has antigen binding properties, a portion thereof is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies. The antibodies against GARS1 protein of the present invention include all antibodies that can be produced by methods known in the art.

Antibodies used for detection of Charcot-Marie-Tooth disease markers of the present invention include intact forms with two full-length light chains and two full-length heavy chains as well as functional fragments of the antibody molecule. A functional fragment of an antibody molecule refers to a fragment that possesses at least an antigen-binding function and includes Fab, F(ab'), F(ab') 2, and Fv.

Additionally, the present invention provides a kit for diagnosing Charcot-Marie-Tooth disease containing the diagnostic composition according to the present invention.

In the present invention, the kit may be a PCR kit, a DNA chip kit, a microarray chip kit, or a protein chip kit, but is not limited thereto.

The PCR kit includes a reaction to amplify the mutation site, examples of which include polymerase chain reaction (PCR), real-time polymerase chain reaction (real-time RCR), and reverse transcription. -Polymerase chain reaction (reverse tranion polymerase chain reaction, RT-PCR), ligase chain reaction (LCR), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182) , transcription-mediated amplification (TMA) (WO88/10315), self sustained sequence replication (20) (WO90/06995), selective amplification of target polynucleotide sequence target polynucleotide sequences, consensus sequence primed polymerase chain reaction (CP-PCR), arbitrarily primed polymerase chain reaction (AP-PCR), and nucleic acid sequence-based amplification (nucleic acid sequence-based amplification). acid sequencebased amplification (NASBA), strand displacement amplification, or loop-mediated isothermal amplification (LAMP) reactions, but are not limited thereto.

For example, the kit for diagnosing Charcot-Marie-Tooth disease of the present invention may include one or more oligonucleotides that specifically bind to a polynucleotide encoding a polypeptide containing a genetic cause mutation of the GARS1 gene. It may include primers corresponding to nucleotides or part of the sequence, reverse transcriptase, Taq polymerase, primers for PCR, and dNTPs, and the analysis method described in relation to "measurement of mRNA expression level" above to measure the polynucleotide expression level. You can use a kit using .

In addition, it may contain metal ion salts such as dNTP (deoxynulceotide triphosphate), heat-resistant polymerase, and magnesium chloride required for PCR reaction, and dNTP, sequencenase, etc. required for sequencing.

In addition, the kit of the present invention is a kit for diagnosing Charcot-Marie-Tooth disease that measures the level of the protein encoded by the GARS1 gene containing the genetic causal mutation, and is a kit for diagnosing Charcot-Marie-Tooth disease by the GARS1 gene containing the genetic causal mutation of the present invention. It may include an antibody that specifically binds to the encoded protein. In addition, the kit for measuring the protein level can be any kit using the above-described method used for “measuring protein expression level”, and is preferably an ELISA kit or a protein chip kit.

Protein expression using antibodies is measured by forming an antigen-antibody complex between a protein encoded by the GARS1 gene containing a genetic mutation and its antibody, and quantitatively by measuring the amount of the complex formed by various methods. can be detected.

In the present invention, the term antigen-antibody complex refers to a combination of a protein encoded by the GARS1 gene containing a genetic causative mutation and an antibody specific for the protein according to the present invention, and the amount of antigen-antibody complex formed is determined by the detection label. Quantitative measurement is possible through the size of the signal (detection label).

In addition, the kit of the present invention includes an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a label that develops color by reaction with a substrate, a chromogenic substrate solution that will undergo color development with the label, a washing solution, and It may contain an enzyme reaction stopping solution, etc., and may be manufactured into a number of separate packaging or compartments containing the reagent components used.

The kit may include tools, reagents, etc. commonly used in the art in addition to the diagnostic composition according to the present invention.

The kits may also take the form of bottles, tubs, sachets, envelopes, tubes, ampoules, etc., which may be partially or entirely made of plastic, glass, paper, foil, wax, etc. It can be formed from etc. The container may be equipped with a completely or partially removable closure that may initially be part of the container or may be attached to the container by mechanical, adhesive, or other means. The container may also be equipped with a stopper, allowing access to the contents by means of a needle. The kit may include an external package, and the external package may include instructions for use of the components.

In addition, the present invention provides a method for treating Charcot-Marie-Tooth disease, comprising the step of detecting a GARS1 mutant gene or a GARS1 mutant protein encoded by the GARS1 mutant gene in a biological sample isolated from a subject. Provides a method of providing information for diagnosis or prediction of disease risk.

In the present invention, the “subject” refers to an individual for whom it is intended to determine whether or not the disease has occurred or is likely to develop Charcot-Marie-Tooth disease, or to predict the risk of developing it. The object may be a vertebrate, and specifically mammals and amphibians. , reptiles, birds, etc., and more specifically, it may be a mammal, for example, a human (Homo sapiens).

In the present invention, “biological sample” refers to any sample obtained from a subject in which the expression of a gene or protein of the present invention can be detected. Preferably, the sample may be one or more selected from the group consisting of biopsy, blood, serum, plasma, lymph, cerebrospinal fluid, ascites, skin tissue, liquid culture, feces, and urine, but is not particularly limited thereto. , it can be prepared by processing by a method commonly used in the technical field of the present invention.

In addition, the detection is for the detection of the GARS1 mutant gene containing the 2,171st mutation site in the nucleotide sequence shown in SEQ ID NO: 1, using reverse transcription polymerase reaction, competitive reverse transcription polymerase reaction, real-time reverse transcription polymerase reaction, and RNase protection assay. (RPA), Northern blotting, sequencing analysis, hybridization by microarray, allele specific PCR (allele specific PCR), dynamic allele-specific hybridization (DASH), PCR extension analysis, It may be performed by one or more methods selected from the group consisting of TaqMan probe PCR analysis and DNA chip, but is not limited thereto.

Among the above methods, sequencing analysis can be performed using a conventional method for determining base sequences and can be performed using an automated genetic analyzer (Sanger, F. et al., Proc. Natl. Acad. Sci. USA. , 74(12), 5463-5467, 1977; Maxam, A. M. and Gilbert, W., Proc. Natl. Acad. Sci. USA., 74(2), 560-564, 1997).

Allele-specific PCR analysis refers to a PCR method that amplifies the DNA fragment where the mutation is located using a primer set including a primer designed with the 3' end of the base where the mutation is located. The principle of the method is that, for example, when a specific base is substituted from G to A, PCR is performed by designing a primer containing the G as the 3' terminal base and a reverse primer capable of amplifying a DNA fragment of an appropriate size. When performing the reaction, if the base at the mutation position is G, the amplification reaction is performed normally and a band at the desired position is observed, and if the base is replaced with A, the primer can complement the template DNA, but 3 ' It takes advantage of the characteristic that the amplification reaction is not performed properly due to the terminal's inability to make complementary bonds (Newton, C. R. et al., Nucleic Acids Res., 17(1), 2503-2516, 1989).

TaqMan probe PCR analysis (Livak, K. J., Genet. Anal., 14, 143-149, 1999) includes 1) designing and manufacturing primers and TaqMan probes to amplify the desired DNA fragment; 2) labeling probes of different alleles with FAM dye and VIC dye (Applied Biosystems, USA); 3) performing PCR using the above primers and probes using the DNA as a template; 4) After the above PCR reaction is completed, analyzing and confirming the TaqMan assay plate with a base sequence analyzer; and 5) determining the genotypes of the polynucleotides of step 1 from the analysis results.

Dynamic allelic hybridization (DASH) analysis can be performed using the method designed by Prince et al. (Prince, J. A. et al., Genome Res. 11(1), 152-162, 2001).

In the PCR extension analysis, a DNA fragment containing the base where the mutation is located is first amplified with a pair of primers, and then all nucleotides added to the reaction are inactivated by dephosphorylation, followed by extension primers specific for the mutation, a dNTP mixture, and DD. This is accomplished by performing a primer extension reaction by adding oxynucleotides, reaction buffer, and DNA polymerase. At this time, the extension primer uses the base immediately adjacent to the 5' direction of the base where the mutation is located as the 3' end, and the dNTP mixture excludes nucleic acids having the same base as the dideoxynucleotide, and the dideoxynucleotide represents the mutation. It is selected from one of the base types. For example, when there is a substitution from G to A, and a mixture of dATP, dCTP and TTP and ddGTP are added to the reaction, the primer is extended by DNA polymerase at the base where the substitution occurred, and after passing a few bases The primer extension reaction is terminated by ddGTP at the position where the G base first appears. If the substitution has not occurred, the extension reaction is terminated at that position, so the type of base representing the mutation can be determined by comparing the lengths of the extended primers.

At this time, the detection method is to detect the mutation by detecting fluorescence using a genetic analyzer (for example, ABI's Model 3700, etc.) used for general base sequence determination in the case of fluorescently labeled extension primers or dideoxynucleotides. (Chen, J., Genome Res., 10(4), 549-557, 2000), and when using unlabeled extension primers and dideoxy nucleotides, matrix assisted laser desorption ionization (MALDI-TOF) The mutation can be detected by measuring the molecular weight using the -time offlight) technique (Ross, P. L., Anal. Chem, 69(20), 4197-4202, 1997).

Alternatively, the detection is for the detection of a protein encoded by the GARS1 mutant gene containing the 2,171st mutation site in the nucleotide sequence shown in SEQ ID NO: 1, and specifically, the 724th mutation site in the amino acid sequence shown in SEQ ID NO: 2. To detect mutation sites, Western blotting, ELISA (Enzymelinked immunosorbent assay), ELLA (enzyme-linked lectin assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, It may be performed by one or more methods selected from the group consisting of immunohistochemical staining, immunoprecipitation analysis, complement fixation analysis, FACS, and protein chip, but is not limited thereto.

In the information provision method of the present invention, if the 2,171st base of the GARS1 gene is a mutation in which C (cytosine) is replaced by A (adenine), the subject is diagnosed as having Charcot-Marie-Tooth disease, or Alternatively, the risk of developing the disease may be predicted to be high.

Additionally, in the information provision method of the present invention, the 2,171st mutation site in the base sequence of the GARS1 gene is replaced with Adenine, whereby the 724th amino acid of the GARS1 protein changes from P (proline) to H (histidine). When replaced with , the subject may be diagnosed as having developed Charcot-Marie-Tooth disease, or may be predicted to have a high risk of developing Charcot-Marie-Tooth disease.

The present invention also provides a screening method for a treatment for Charcot-Marie-Tooth disease comprising the following steps:

(a) treating target cells with a candidate drug for the treatment of Charcot-Marie-Tooth disease;

(b) measuring whether a GARS1 mutant gene or a GARS1 mutant protein encoded by the GARS1 mutant gene is detected in the cells of step (a); and

(c) If the GARS1 mutant gene or GARS1 mutant protein is not detected in the cells measured in step (b), determining it as a treatment for Charcot-Marie-Tooth disease.

In the above screening method, “candidate” refers to an unknown test substance used in screening to test whether it affects the expression level of a gene or affects the expression or activity of a protein. The candidate substances include, for example, compounds, nucleotides, proteins, antibodies, and natural product extracts, but are not limited thereto.

Detection of the GARS1 mutant gene or the GARS1 mutant protein encoded by the GARS1 mutant gene may be performed in the same manner as the detection method of the GARS1 mutant gene or the GARS1 mutant protein encoded by the GARS1 mutant gene described above, but is limited thereto. It doesn't work.

The contents of the present invention described above are applied equally to each other unless they contradict each other, and implementation by a person skilled in the art with appropriate changes is also included in the scope of the present invention.

Hereinafter, the present invention will be described in detail through examples, but the scope of the present invention is not limited to the following examples.

Experimental Example 1. Cohort study preparation

This study was conducted on a cohort of 1,064 unrelated Korean CMT cases (families) with CMT ancestry, including 1,230 patients with CMT neuropathy and 200 healthy controls. As a result of copy number variation analysis of the 17p12 region, 354 cases were confirmed to be CMT1A overlapping PMP22, and 710 cases were additionally investigated to confirm ARS gene mutations. Written consent was obtained from all patients, and the procedure was approved by the institutional review boards of Sungkyunkwan University, Samsung Seoul Hospital (2014-08-057-002), and Kongju National University (KNU_IRB_2018-06).

Experimental Example 2. Clinical assessment

Clinical information on motor and sensory impairment, deep tendon reflexes and muscle atrophies was obtained in a standardized manner. The strength of flexor and extensor muscles was assessed manually using standard Medical Research Council (MRC) scales. Physical disability was measured by two scales: the functional disblility scale (FDS) and the CMT neuropathy score version 2 (CMTNSv2). Sensory disturbances were assessed by pain severity and level, temperature, vibration, and posture. Age of onset was determined by asking patients at what age symptoms of distal muscle weakness, foot deformity, or sensory changes first occurred.

Experimental Example 3. Electrophysiological examinations

Motor and sensory conduction velocities of the median, ulnar, radial, peroneal, tibial, and sural nerves were surface stimulated and recorded. Determination was made using a standard method using a recording electrode. Distal terminal latencies (DTLs), motor nerve conduction velocities (MNCVs), and compound muscle action potentials (CMAPs) of the distal ends of the median, ulnar, and radial nerves at the elbow and wrist. The decision was made by stimulation. Fibula and tibia MNCVs and CMAPs were determined by stimulating the knee and ankle. CMAP amplitude was measured from baseline to negative peak values. Sensory nerve conduction velocities (SNCVs) were measured using the orthodromic method from the median, ulnar, and radial nerves across the finger-wrist segment, as well as over the sural nerve.

Experimental Example 4. Molecular genetic study DNA purification and paternity test

Genomic DNA was purified from whole blood using the HiGene Genomic DNA extraction kit (Biofact). The exome was obtained using the SureSelect Human ALL Exon 50M kit (Agilent Technologies, Santa Clara), and sequencing was performed using the HiSeq 2000 genome analyzer (Illumina, San Diego). Genome acquisition and sequencing were performed at Macrogen Inc., and UCSC assembly hg19 (GRCh37) was used as a reference sequence. Small nucleotide variants (SNVs) were identified using GATK and SAMtools programs. Rare or novel alleles with a frequency of less than 0.1 were identified using the 1000 Genomes Project, Genome Aggregation database, and Korean Reference Genome Database (KRGDB). The pathogenicity of SNV was evaluated in five grades according to the guidelines of the American College of Medical Genetics and Genomics (ACMG). Pathogenic candidate variants were confirmed through Sanger sequencing using a SeqStudio genetic analyzer (Life Technologies-Thermo Fisher Scientific).

Experimental Example 5. In silico prediction and conservation analysis

In silico analysis to predict mutation effects was performed using MUpro, PolyPhen-2, PROVEAN, and SIFT. The preservation of the mutation site was analyzed using MEGA-X version 5.05. Genomic evolutionary rate profiling score (GERP) was determined using the GERP++ program. For protein 3D structure prediction and modeling, I-TASSER was used and visualization was performed using the Mol* function of the Protein data bank.

Experimental Example 6. Statistical analysis

Clinical characteristics of patients according to ARS genes were compared using non-parametric Kruskal-Wallis test and post hoc Dunn's multiple comparison. Missing values of clinical characteristics were excluded from the analysis. Parametric PeARSon r or non-parametric Spearman r were used to analyze the correlation between severity and age of onset. It was considered statistically significant at p<0.05, and all statistical analyzes and calculations were performed using GraphPad Prism ver.8.00.

Example 1. Identification of causative mutations in the ARS gene

As a result of the cohort study, 11 pathogenic or pathogenic mutations (13 families) were identified in four ARS genes, as shown in Table 1 below.

In Table 1, 1000G (1000 Genomes database), ACMG (American College of Medical Genetics and Genomics), GERP (genomic evolutionary rate profiling score), gnomAD (Genome Aggregation Database), KRGDB (Korean Reference Genome Database), LP ( It means likely pathogenic, NR (not reported), and P (pathogenic).

Reference nucleotide and amino acid sequences are as follows (GenBank accession numbers): GARS1 : NM_002047.4 and NP_002038.2, HARS1 : NM_002109.6 and NP_002100.2, WARS1 : NM_004184.4 and NP_004175.2, YARS1 : NM_003680.4 and NP_003671.1.

In silico analysis: scores of PROVEAN (PRO) <-2.5, PolyPhen-2 (PP2) ~1, MUpro (MUp) <0, and SIFT <0.05 indicate pathogenicity prediction (* indicates pathogenicity prediction).

The genotypes of all pedigree members tested are shown in Figure 1, and all of these causative mutations were confirmed through Sanger sequencing (Figure 2). The amino acids of most mutations were highly conserved from yeast to vertebrates, and the mutation site was located in the important aminoacyl-tRNA synthetase (catalytic) domain (Figure 3). Four GARS1 mutations were observed in four pedigrees. In addition, two YARS1 mutations were observed in three families (Figure 1D) and two mutations were found in each of HARS1 and WARS1 in each of the two families (Figures 1B and 1C).

The four pathogenic mutations identified in CMT2 or GARS1 in the dHMN family are as follows: c.598G>A (p.D200N) in the dHMN family (FC377), and c.794C>T (p.S265F) in the dHMN family (FC145). ), c.1007C>A (p.P336H) in the large dHMN family (FC825) and c.2171C>A (p.P724H) in the CMT2 family (FC855) were identified (Figure 1A). In the above, p.D200N and p.S265F have been reported previously, but other mutations have been newly reported through the present invention. In particular, in the FC500 family with p.P724H, both unaffected parents did not show the mutation, so it was judged to be a de novo mutation.

Example 2. In silico prediction of protein structure

The pathogenic effects of the gene mutations in Table 1 above were predicted through in silico analysis using the PROVEAN, PolyPhen-2 (PP2), MUpro, and SIFT programs. For novel or potentially pathogenic mutations, 3D conformational changes were simulated by the I-TASSER and Mol* functions of the Protein Databank (Figure 4).

GARS1 wild type (p.P336) formed a hydrogen bond between p.S338 and p.R342. However, p.S338 was predicted to bind to p.R337, causing modification of the β-sheet at residues 339-343 of the p.P336H mutation. In the p.P724H mutant, wild-type GARS1 formed p.P724 and p.R696 and p.Q694 and p.L725. In the P724H mutation, it was predicted that both hydrogen bonds would be destroyed and a new bond would be created between p.H724 and p.L725, moving away from the anticodon domain.

Example 3. Clinical features of CMT patients with ARS gene mutations

The clinical features of 17 CMT patients (9 GARS1 , 2 HARS1, 2 WARS1 , and 4 YARS1 ) with ARS gene mutations were confirmed and summarized in Table 2 below.

In Table 2, AOE (age of examination), AOO (age of onset), CMT (Charcot-Marie-Tooth disease), CMTNS (CMT neuropathy score) (CMT neuropathy score), dHMN (distal hereditary motor neuropathy), FDS (functional disability scale), ND (Not done). a+ means MRC (medical Intrinsic hand weakness is 4/5 on the research council scale, a++ is intrinsic hand weakness <4/5 on the MCR scale, a+++ is proximal weakness, and a- means no symptoms. b+ means ankle dorsiflexion 4/5 on the MRC scale, b++ means dorsiflexion <4/5 on the MCR scale, b+++ means proximal weakness, c+ means decreased, and c++ means normal reflexes. reflex), c+++ means hyper reflex, and c- means absence.

The average age of onset was 17.4 ± 10 years, with slight differences depending on the gene. Among the 20 patients, the WARS1 group showed the latest age of onset at 28.0 ± 11.3 years, and the average FDS and CMTNSv2 were determined to be 2.1 ± 1.0 and 10.2 ± 4.9, respectively. In both FDS and CMTNSv2, the GARS1 group showed the most severe symptoms, and the WARS1 group showed the mildest symptoms. All patients in the HARS1 group and two-thirds of the GARS1 patients showed similar degrees of disease disability between the upper and lower extremities. Additionally, hyperreflexia in the knee jerk was observed in 7 patients (35%), a very high rate compared to other CMT patients. In addition, length-dependent sensory loss was observed in 12 patients (60%), and vibration sense was decreased compared to pain. In particular, hand muscle atrophy was frequently observed in GARS1 patients. Pes cavus was found in 15 of 20 patients, and 16 patients showed steppage gait. However, all patients with WARS1 mutations did not show cavus or drooping gait.

In addition, clinical phenotypes were compared between ARS gene groups ( GARS1 and YARS1 ) (Figure 5). There was no difference in test age between each group. Additionally, no differences between each group were identified in FDS, CMTNSv2, median MNCV, and knee and leg muscle weakness. As a result of analyzing the correlation between age of onset and clinical phenotype for all patients with ARS gene mutations, it was confirmed that age of onset had a weak correlation with mild symptoms in terms of severity (Figures 6A and 6B) (CMTNS: r = -0.211, p = 0.385, FDS: r = -0.077, p = 0.747).

Example 4. Electrophysiological findings according to ARS gene mutation

Electrophysiological results of 15 patients (7 GARS1 , 2 HARS1 , 2 WARS1 , and 4 YARS1 ) are shown in Table 3.

In Table 3, A (absent), CMAP (compound muscle action potential), CMT (Charcot-Marie-Tooth disease), and DTL (distal terminal latency), motor nerve conduction velocity (MNCV), sensory nerve action potential (SNAP), and sensory nerve conduction velocity (SNCV). Normal motor DTLs (ms) are <3.6 (median), < 2.5 (ulnar), < 4.8 (peroneal), and < 5.1 (tibial), and normal motor NCVs (m/s) were ≥ 50.5 (median), ≥ 51.1 (ulnar), and ≥ 41.2 (peroneal). ), and ≥ 41.1 (tibial), normal sensory NCVs (m/s) are ≥ 39.3 (median), ≥ 37.5 (ulnar), and ≥ 32.1, and normal motor amplitudes ( Normal sensory amplitudes (mV) are ≥ 6 (median), ≥ 8 (ulnar), ≥ 1.6 (peroneal), and ≥ 6 (tibial), and normal sensory amplitudes (mV) are ≥ 8.8 (median), ≥ 7.9 ( ulnar), and ≥ 6.0 (sural). Abnormal values are indicated in bold.

In patients with GARS1 mutations, DTLs, MNCV, and CMAP measurements in motor neurons generally showed patterns consistent with typical CMT2, which were outside the normal range. Patient FC855 (III-1) showed abnormal electrophysiological findings in the median nerve, ulnar nerve, peroneal nerve, tibial nerve, and sural nerve. Patients with WARS1 mutations were reported to have no sensory nerve conduction abnormalities, but in the present invention, both patients with WARS1 mutations showed abnormal electrophysiological findings in sensory nerves. Patient FC862(II-1) showed abnormal SNCV and SNAP in the median, ulnar, and sural nerves, and patient FC885(II-1) showed abnormal findings in the ulnar and sural nerves. Interestingly, all ARS patients showed abnormal ulnar nerve DTLs. In addition, all but one patient showed abnormal findings in peroneal nerve DTLs. Needle electromyography is compatible with neuropathy based on fibrillation potential and neurogenic motor unit action potential. In this regard, the correlation between age of onset and electrophysiological findings was analyzed for all ARS patients (Figures 6C to 6F). For nerve conduction, age of onset was significantly different for motor median nerve (CMAP: r = 0.371, p = 0.129; MNCV: r = 0.286, p = 0.250) and sensory median nerve (SNAP: r = 0.203, p = 0.419; SNCV: r = 0.216, p = 0.390), it was confirmed that there was a slight correlation with the increase in speed or potential.

Overall, the present invention identifies mutations in a specific gene through a cohort study of Charcot-Marie-Tooth disease patients, and confirms that the mutation is the causative gene in Charcot-Marie-Tooth disease, and the GARS1 gene according to the present invention The novel c.2171C>A mutation has been confirmed to be the causative gene for Charcot-Marie-Tooth disease, and can be usefully used as a marker for diagnosing Charcot-Marie-Tooth disease.

<110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION KONGJU NATIONAL UNIVERSITY INDUSTRY-UNIVERSITY COOPERATION FOUNDATION Research and Business Foundation SUNGKYUNKWAN UNIVERSITY Dong-A University Research Foundation For Industry-Academy Cooperation <120> A novel Charcot-Marie-Tooth disease diagnostic biomarker comprising a mutant of the glycyl-tRNA Synthetase 1 gene and Uses Thereof <130> 1-78P <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 2393 <212> DNA <213> Artificial Sequence <220> <223> glycyl-tRNA Synthetase 1 (GARS1) <400> 1 atgccctctc cgcgtccagt gctgcttaga ggtgctcgcg ccgctctgct gctgctgctg 60 ccgccccggc tcttagcccg accctcgctc ctgctccgcc ggtccctcag cgcggcctcc 120 tgccccccga tctccttgcc cgccgccgcc tcccggagca gcatggacgg cgcggggggct 180 gaggaggtgc tggcacctct gaggctagca gtgcgccagc agggagatct tgtgcgaaaa 240 ctcaaagaag ataaagcacc ccaagtagac gtagacaaag cagtggctga gctcaaagcc 300 cgcaagaggg ttctggaagc aaaggagctg gcgttacagc ccaaagatga tattgtagac 360 cgagcaaaaa tggaagatac cctgaagagg aggtttttct atgatcaagc ttttgctatt 420 tatggaggtg ttagtggtct gtatgacttt gggccagttg gctgtgcttt gaagaacaat 480 attattcaga cctggaggca gcactttatc caagaggaac agatcctgga gatcgattgc 540 accatgctca cccctgagcc agttttaaag acctctggcc atgtagacaa atttgctgac 600 ttcatggtga aagacgtaaa aaatggagaa tgttttcgtg ctgaccatct attaaaagct 660 catttacaga aattgatgtc tgataagaag tgttctgtcg aaaagaaatc agaaatggaa 720 agtgttttgg cccagcttga taactatgga cagcaagaac ttgcggatct ttttgtgaac 780 tataatgtaa aatctcccat tactggaaat gatctatccc ctccagtgtc ttttaactta 840 atgttcaaga ctttcattgg gcctggagga aacatgcctg ggtacttgag accagaaact 900 gcacagggga ttttcttgaa tttcaaacga cttttggagt tcaaccaagg aaagttgcct 960 tttgctgctg cccagattgg aaattctttt agaaatgaga tctcccctcg atctggactg 1020 atcagagtca gagaattcac aatggcagaa attgagcact ttgtagatcc cagtgagaaa 1080 gaccacccca agttccagaa tgtggcagac cttcaccttt atttgtattc agcaaaagcc 1140 caggtcagcg gacagtccgc tcggaaaaatg cgcctgggag atgctgttga acagggtgtg 1200 attaataaca cagtattagg ctatttcatt ggccgcatct acctctacct cacgaaggtt 1260 ggaatatctc cagataaact ccgcttccgg cagcacatgg agaatgagat ggcccattat 1320 gcctgtgact gttgggatgc agaatccaaa acatcctacg gttggattga gattgttgga 1380 tgtgctgatc gttcctgtta tgacctctcc tgtcatgcac gagccaccaa agtcccactt 1440 gtagctgaga aacctctgaa agaacccaaaa acagtcaatg ttgttcagtt tgaacccagt 1500 aagggagcaa ttggtaaggc atataagaag gatgcaaaac tggtgatgga gtatcttgcc 1560 atttgtgatg agtgctacat tacagaaatg gagatgctgc tgaatgagaa aggggaattc 1620 acaattgaaa ctgaagggaa aacatttcag ttaacaaaag acatgatcaa tgtgaagaga 1680 ttccagaaaa cactatatgt ggaagaagtt gttccgaatg taattgaacc ttccttcggc 1740 ctgggtagga tcatgtatac ggtatttgaa catacattcc atgtacgaga aggagatgaa 1800 cagagaacat tcttcagttt ccctgctgta gttgctccat tcaaatgttc cgtcctccca 1860 ctgagccaaa accaggagtt catgccattt gtcaaggaat tatcggaagc cctgaccagg 1920 catggagtat ctcacaaagt agacgattcc tctgggtcaa tcggaaggcg ctatgccagg 1980 actgatgaga ttggcgtggc ttttggtgtc accattgact ttgacacagt gaacaagacc 2040 ccccacactg caactctgag ggaccgtgac tcaatgcggc agataagagc agagatctct 2100 gagctgccca gcatagtcca agacctagcc aatggcaaca tcacatgggc tgatgtggag 2160 gccaggtatc ctctgtttga agggcaagag actggtaaaa aagagacaat cgaggaatga 2220 ggacaatttt gacaactttt gaccacttgc gctaataaaa aaaaaaaaaaa actactctta 2280 tgtccacttt acaaaagaaa acagcattgt gattactccc agggaccgta ttttatcttc 2340 agtggctgcc tgattttacc cccacaatta aagttgaagg aatcctgaac aaa 2393 <210> 2 <211> 739 <212> PRT <213> Artificial Sequence <220> <223> glycyl-tRNA Synthetase 1 (GARS1) <400> 2 Met Pro Ser Pro Arg Pro Val Leu Leu Arg Gly Ala Arg Ala Ala Leu 1 5 10 15 Leu Leu Leu Leu Pro Pro Arg Leu Leu Ala Arg Pro Ser Leu Leu Leu 20 25 30 Arg Arg Ser Leu Ser Ala Ala Ser Cys Pro Pro Ile Ser Leu Pro Ala 35 40 45 Ala Ala Ser Arg Ser Ser Met Asp Gly Ala Gly Ala Glu Glu Val Leu 50 55 60 Ala Pro Leu Arg Leu Ala Val Arg Gln Gln Gly Asp Leu Val Arg Lys 65 70 75 80 Leu Lys Glu Asp Lys Ala Pro Gln Val Asp Val Asp Lys Ala Val Ala 85 90 95 Glu Leu Lys Ala Arg Lys Arg Val Leu Glu Ala Lys Glu Leu Ala Leu 100 105 110 Gln Pro Lys Asp Asp Ile Val Asp Arg Ala Lys Met Glu Asp Thr Leu 115 120 125 Lys Arg Arg Phe Phe Tyr Asp Gln Ala Phe Ala Ile Tyr Gly Gly Val 130 135 140 Ser Gly Leu Tyr Asp Phe Gly Pro Val Gly Cys Ala Leu Lys Asn Asn 145 150 155 160 Ile Ile Gln Thr Trp Arg Gln His Phe Ile Gln Glu Glu Gln Ile Leu 165 170 175 Glu Ile Asp Cys Thr Met Leu Thr Pro Glu Pro Val Leu Lys Thr Ser 180 185 190 Gly His Val Asp Lys Phe Ala Asp Phe Met Val Lys Asp Val Lys Asn 195 200 205 Gly Glu Cys Phe Arg Ala Asp His Leu Leu Lys Ala His Leu Gln Lys 210 215 220 Leu Met Ser Asp Lys Lys Cys Ser Val Glu Lys Lys Ser Glu Met Glu 225 230 235 240 Ser Val Leu Ala Gln Leu Asp Asn Tyr Gly Gln Gln Glu Leu Ala Asp 245 250 255 Leu Phe Val Asn Tyr Asn Val Lys Ser Pro Ile Thr Gly Asn Asp Leu 260 265 270 Ser Pro Pro Val Ser Phe Asn Leu Met Phe Lys Thr Phe Ile Gly Pro 275 280 285 Gly Gly Asn Met Pro Gly Tyr Leu Arg Pro Glu Thr Ala Gln Gly Ile 290 295 300 Phe Leu Asn Phe Lys Arg Leu Leu Glu Phe Asn Gln Gly Lys Leu Pro 305 310 315 320 Phe Ala Ala Ala Gln Ile Gly Asn Ser Phe Arg Asn Glu Ile Ser Pro 325 330 335 Arg Ser Gly Leu Ile Arg Val Arg Glu Phe Thr Met Ala Glu Ile Glu 340 345 350 His Phe Val Asp Pro Ser Glu Lys Asp His Pro Lys Phe Gln Asn Val 355 360 365 Ala Asp Leu His Leu Tyr Leu Tyr Ser Ala Lys Ala Gln Val Ser Gly 370 375 380 Gln Ser Ala Arg Lys Met Arg Leu Gly Asp Ala Val Glu Gln Gly Val 385 390 395 400 Ile Asn Asn Thr Val Leu Gly Tyr Phe Ile Gly Arg Ile Tyr Leu Tyr 405 410 415 Leu Thr Lys Val Gly Ile Ser Pro Asp Lys Leu Arg Phe Arg Gln His 420 425 430 Met Glu Asn Glu Met Ala His Tyr Ala Cys Asp Cys Trp Asp Ala Glu 435 440 445 Ser Lys Thr Ser Tyr Gly Trp Ile Glu Ile Val Gly Cys Ala Asp Arg 450 455 460 Ser Cys Tyr Asp Leu Ser Cys His Ala Arg Ala Thr Lys Val Pro Leu 465 470 475 480 Val Ala Glu Lys Pro Leu Lys Glu Pro Lys Thr Val Asn Val Val Gln 485 490 495 Phe Glu Pro Ser Lys Gly Ala Ile Gly Lys Ala Tyr Lys Lys Asp Ala 500 505 510 Lys Leu Val Met Glu Tyr Leu Ala Ile Cys Asp Glu Cys Tyr Ile Thr 515 520 525 Glu Met Glu Met Leu Leu Asn Glu Lys Gly Glu Phe Thr Ile Glu Thr 530 535 540 Glu Gly Lys Thr Phe Gln Leu Thr Lys Asp Met Ile Asn Val Lys Arg 545 550 555 560 Phe Gln Lys Thr Leu Tyr Val Glu Glu Val Val Pro Asn Val Ile Glu 565 570 575 Pro Ser Phe Gly Leu Gly Arg Ile Met Tyr Thr Val Phe Glu His Thr 580 585 590 Phe His Val Arg Glu Gly Asp Glu Gln Arg Thr Phe Phe Ser Phe Pro 595 600 605 Ala Val Val Ala Pro Phe Lys Cys Ser Val Leu Pro Leu Ser Gln Asn 610 615 620 Gln Glu Phe Met Pro Phe Val Lys Glu Leu Ser Glu Ala Leu Thr Arg 625 630 635 640 His Gly Val Ser His Lys Val Asp Asp Ser Ser Gly Ser Ile Gly Arg 645 650 655 Arg Tyr Ala Arg Thr Asp Glu Ile Gly Val Ala Phe Gly Val Thr Ile 660 665 670 Asp Phe Asp Thr Val Asn Lys Thr Pro His Thr Ala Thr Leu Arg Asp 675 680 685 Arg Asp Ser Met Arg Gln Ile Arg Ala Glu Ile Ser Glu Leu Pro Ser 690 695 700 Ile Val Gln Asp Leu Ala Asn Gly Asn Ile Thr Trp Ala Asp Val Glu 705 710 715 720 Ala Arg Tyr Pro Leu Phe Glu Gly Gln Glu Thr Gly Lys Lys Glu Thr 725 730 735 Ile Glu Glu

Claims (18)

A biomarker composition for diagnosing Charcot-Marie-Tooth disease (CMT), comprising a genetic causal mutation in the Glycyl-tRNA Synthetase 1 ( GARS1 ) gene.
According to clause 1,
The mutation is a biomarker composition, characterized in that the 2,171st base of the GARS1 gene represented by SEQ ID NO: 1 is substituted from C (cytosine) to A (adenine).
A biomarker for diagnosing Charcot-Marie-Tooth disease (CMT), comprising a GARS1 mutant protein encoded by a GARS1 mutant gene containing the 2,171st mutation site in the base sequence shown in SEQ ID NO: 1. Composition.
According to clause 3,
The GARS1 mutant protein is a mutant protein encoded by a gene in which the 2,171st base of the GARS1 gene, shown in SEQ ID NO: 1, is substituted from C (cytosine) to A (adenine), and is an amino acid sequence shown in SEQ ID NO: 2. A biomarker composition, characterized in that the 724th amino acid is substituted from P (proline) to H (histidine).
Charcot-Marie-Tooth disease, comprising an agent capable of detecting a GARS1 mutant gene containing the 2,171st mutation site in the base sequence shown in SEQ ID NO: 1 or a GARS1 mutant protein encoded by the GARS1 mutant gene. -Marie-Tooth disease (CMT) diagnostic composition.
According to clause 5,
A diagnostic composition, characterized in that Charcot-Marie-Tooth disease is diagnosed when the 2,171st mutation site in the base sequence of the GARS1 gene represented by SEQ ID NO: 1 is adenine.
According to clause 5,
A diagnostic composition, characterized in that Charcot-Marie-Tooth disease is diagnosed when the 724th amino acid in the amino acid sequence of the GARS1 protein represented by SEQ ID NO: 2 is H (histidine).
According to clause 5,
The agent is a group consisting of a primer, a probe, and an anti-sense nucleotide that specifically binds to the GARS1 mutant gene mRNA containing the 2,171st mutation site in the base sequence shown in SEQ ID NO: 1. A diagnostic composition, characterized in that it is one or more selected from the following.
According to clause 5,
The agent is an oligopeptide , monoclonal antibody, polyclonal antibody, chimeric ( A diagnostic composition, characterized in that it is one or more selected from the group consisting of a chimeric antibody, a ligand, a peptide nucleic acid (PNA), and an aptamer.
A kit for diagnosing Charcot-Marie-Tooth disease, comprising the diagnostic composition of any one of claims 5 to 9.
According to clause 10,
A diagnostic kit, characterized in that the kit is a PCR kit, DNA chip kit, microarray chip kit, or protein chip kit.
Diagnosis or risk of developing Charcot-Marie-Tooth disease, comprising detecting a GARS1 variant gene or a GARS1 variant protein encoded by the GARS1 variant gene in a biological sample isolated from the subject. How to provide information for forecasting.
According to clause 12,
Information, wherein the biological sample is at least one selected from the group consisting of saliva, biopsy, blood, serum, plasma, lymph, cerebrospinal fluid, ascites, skin tissue, liquid culture, feces, and urine. How to provide.
According to clause 12,
In the GARS1 gene, if the 2,171st base in the nucleotide sequence of the GARS1 gene represented by SEQ ID NO: 1 is a mutation in which C (cytosine) is replaced by A (adenine), the subject has Charcot-Marie-Tooth disease. A method of providing information characterized by diagnosing a target or predicting a high risk of developing the disease.
According to clause 12,
In the GARS1 protein, if the 724th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is a mutation in which P (proline) is replaced with H (histidine), the subject is diagnosed as having Charcot-Marie-Tooth disease. A method of providing information, characterized by predicting a high risk of developing the disease.
According to clause 12,
The gene detection is performed using reverse transcription polymerase reaction, competitive reverse transcription polymerase reaction, real-time reverse transcription polymerase reaction, RNase protection assay (RPA), Northern blotting, sequencing analysis, hybridization by microarray, and allele-specific PCR ( Provide information, characterized in that it is performed by one or more methods selected from the group consisting of allele specific PCR), dynamic allele-specific hybridization (DASH), PCR extension analysis, TaqMan probe PCR analysis, and DNA chip. method.
According to clause 12,
The protein detection is performed by Western blotting, ELISA (Enzymelinked immunosorbent assay), ELLA (enzyme-linked lectin assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, and immunohistochemistry. A method of providing information, characterized in that it is performed by one or more methods selected from the group consisting of staining, immunoprecipitation analysis, complement fixation analysis, FACS, and protein chip.
A method for screening a therapeutic agent for Charcot-Marie-Tooth disease comprising the following steps:
(a) treating target cells with a candidate drug for the treatment of Charcot-Marie-Tooth disease;
(b) measuring whether a GARS1 mutant gene or a GARS1 mutant protein encoded by the GARS1 mutant gene is detected in the cells of step (a); and
(c) If the GARS1 mutant gene or GARS1 mutant protein is not detected in the cells measured in step (b), determining it as a treatment for Charcot-Marie-Tooth disease.