KR101967880B1 - Marker for diagnosing Charcot-Marie-Tooth disease and use thereof - Google Patents

Abstract

The HSPB3 protein mutant or HSPB3 mutant according to one aspect of the present invention can be used as a marker for diagnosing Charcot-Marie-Tooth disease. The diagnosis using the HSPB3 gene mutant enables accurate early diagnosis of Charcot-Marie-Tooth disease through genetic testing, thereby maximizing the therapeutic effect by applying the recently developed treatment method early.

Description

Marker for diagnosing Charcot-Marie-Tooth disease and use thereof

HSPB3 mutant or HSPB3 mutant, a composition capable of specifically detecting these mutants, and a kit for diagnosing Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) or hereditary motor and sensory neuropathy is a disorder of the hereditary and clinically heterogeneous peripheral nervous system, with loss of sensory function And weakness of the original satellite muscle (Harding et al., Brain 103: 259-280 (1980)). CMT is the most common type of genetic peripheral neuropathy. It is associated with more than 80 causative genes. According to histopathological and electrophysiologic results, CMT was (1) dehydrated CMT1 showing a median motor nerve conduction velocity of 38 m / s or less; (2) axon indicating a median nerve conduction velocity greater than 38 m / s CMT2; And (3) a median IntCMT that exhibits a median motor nerve conduction velocity between 25 and 45 m / s and exhibits neuropathology characterized by axonal and / or dehydration characteristics.

The incidence of CMT is high among rare diseases inherited by one person per 2,500 people. In CMT patients, the muscles of the feet and hands gradually contract and weaken, deforming the shape of the foot and hands, and causing facial paralysis and hearing impairment. The onset usually occurs around the age of 10, but rarely after the age of 30. Patients' symptoms vary from mild to near normal, depending on the type of gene mutation, to the extent that they need help with walking or are dependent on a wheelchair.

There was a report in which transgenic mice treated with onapristone, an antagonist of progesterone receptor, decreased the overexpression of HSPB3 mRNA and improved the phenotype of genetic kinetic neuropathy without side effects, Ascorbic acid, an essential substance in herbal formulations, improved the phenotype of herpes reparative and hereditary kinetic neuropathy in CMT1A transgenic mice and neurotrophin-3 (NT-3) in the CMT1A patient group It was reported that the effect of improving the sensory symptoms was increased by increasing the herniated nerve fibers. Thus, CMT can be used as an adjunct to the genetic diagnosis of genetic neuropathy. However, the development of effective diagnostic methods for hereditary neurological diseases is still insufficient. For the treatment of kinematic neuropathy with very heterogeneous genetic characteristics, it is required to establish accurate genetic diagnosis method first.

Accordingly, the inventors of the present invention have made efforts to investigate the cause of CMT, and as a result, they have uncovered a novel mutant gene which has not been previously reported, and confirmed that the mutant gene can be usefully used for the diagnosis of CMT, Respectively.

One aspect provides heat shock protein family B member 3 (HSPB3) protein variants or polynucleotides encoding the same.

Another aspect provides a composition for diagnosing the onset or the risk of the onset of Charcot-Marie-Tooth disease.

Another aspect provides a kit for diagnosing the onset or the risk of the onset of Charcot-Marie-Tooth disease comprising said composition.

Another aspect provides a method of diagnosing Charcot-Marie-Tooth disease or providing information for predicting the risk of onset.

One aspect is a heat shock protein family member 3 (HSPB3) protein variant consisting of an amino acid sequence in which the tyrosine (Y) at position 118 in the amino acid sequence of SEQ ID NO: 1 is substituted with histidine (H) Lt; / RTI >

The HSPB3 protein variant may have an amino acid mutation in the alpha-cristalline domain of the HSPB3 protein.

The HSPB3 protein variant may be a separate HSPB3 protein variant.

HSPB3 (heat shock protein family B member 3) protein, also known as heat shock 27 kDa protein 3, affects the refolding of denatured proteins as ATP-dependent chaperone proteins and can act as an inhibitor in actin polymerization. The HSPB3 protein may be a protein registered with the NCBI Accession number NP_006299.1, for example, a protein comprising the amino acid sequence represented by SEQ ID NO: 1.

The term "HSPB3 protein variant" means that a mutation has occurred in a part of the amino acid sequence of the HSPB3 protein represented by SEQ ID NO: 1. Specifically, it may be a protein encoded by a HSPB3 gene mutant in which the thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 is replaced with cytosine (C), and more specifically, the amino acid sequence represented by SEQ ID NO: 1 May be a HSPB3 protein variant in which tyrosine (Y) at position 118 in position is replaced with histidine (H) (p.Tyr118His or p.Y118H).

Another aspect provides a polynucleotide encoding a heat shock protein family member 3 (HSPB3) protein variant.

The polynucleotide encoding the HSPB3 protein variant may be one encoding an HSPB3 protein variant having an amino acid mutation in the alpha-cristalline domain of the HSPB3 protein.

The polynucleotide encoding the HSPB3 protein variant may be a polynucleotide consisting of a nucleotide sequence in which the thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 is replaced with a cytosine (C). Thus, the HSPB3 protein variant is encoded by a polynucleotide consisting of a nucleotide sequence in which the thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 has been replaced by a cytosine (C) and is located at position 118 in the amino acid sequence of SEQ ID NO: Of the tyrosine (Y) is replaced with histidine (H).

The HSPB3 ( heat shock protein family B member 3) gene may be a gene encoding the HSPB3 protein, and may be a gene registered with NCBI Accession number NM_006308.2. For example, a gene containing the nucleotide sequence represented by SEQ ID NO: 2 Lt; / RTI >

The term " HSPB3 gene variant" refers to HSPB3 Means that a mutation has occurred in a part of the nucleotide sequence of the gene. Specifically, it may be a HSPB3 gene mutant in which the thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 is replaced by a cytosine (C) (c.352T> C). Thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 may be a sequence position that is well conserved among vertebrates.

The HSPB3 The gene mutant is HSPB3 It may be a point mutation of a gene, specifically a missense mutation.

Those skilled in the art will readily be able to identify the position, nucleotide sequence and amino acid sequence of the mutation using the registration number. The specific sequence corresponding to the number registered in the UCSC genome browser or GenBank may change over time. It will be apparent to those of ordinary skill in the art that the scope of the present invention also affects the altered sequence.

An HSPB3 protein variant consisting of an amino acid sequence in which the tyrosine (Y) at position 118 in the amino acid sequence of SEQ ID NO: 1 is replaced with histidine (H), or a mutant at position 352 in the nucleotide sequence of SEQ ID NO: The HSPB3 gene mutant consisting of the nucleotide sequence substituted with the nucleotide sequence (C) can be a diagnostic marker of Charcot-Marie-Tooth disease. Thus, the p.Y118H mutation site of the HSPB3 protein mutant or the c.352T > C mutation site of the HSPB3 mutant may be a mutation marker for the diagnosis of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is a disease caused by a mutation of a gene involved in peripheral nerve development of the hands and feet. It is a type 1 , Type 2 associated with axonal abnormalities, and type 3 and type 4 associated with type 1 and type 2, and type X associated with the X chromosome.

Charcot-Marie-Tooth Disease type 2 (CMT2) is an autosomal dominant axon neuropathy. It has a greater degree of impairment or age at onset than CMT1, and the nerve conduction velocity is normal or slightly lower . Specifically, the HSPB3 protein variant or HSPB3 gene variant may be a diagnostic marker of CMT2.

The term "diagnosis" means identifying the presence or characteristic of a pathological condition. Therefore, the above-mentioned "diagnosis of CMT" may refer to whether or not the CMT is caused or to predict the onset of the onset of CMT.

The term "diagnosis marker" may refer to a substance capable of diagnosing the onset or susceptibility of the CMT.

Selection and application of significant diagnostic markers can determine the reliability of diagnostic results. Significant diagnostic markers may be markers with high reliability with high validity and consistency in repeated measurements. An HSPB3 protein variant consisting of an amino acid sequence in which the tyrosine (Y) at position 118 in the amino acid sequence of SEQ ID NO: 1 is substituted with histidine (H) as the above diagnostic marker, or a mutant of the TIN at position 352 in the nucleotide sequence of SEQ ID NO: ) Mutant of the HSPB3 gene consisting of the nucleotide sequence substituted by the cytosine (C) is highly reliable marker that is detected only in the CMT patient and is rarely detected in the normal control group. Therefore, the diagnosis result based on the result obtained by detecting the presence or absence of the mutant can be reasonably reliable.

Another aspect is a protein consisting of an amino acid sequence in which tyrosine (Y) at position 118 in SEQ ID NO: 1 is substituted with histidine (H), or a protein in which the thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: And a preparation capable of specifically detecting a polynucleotide consisting of a nucleotide sequence substituted by the nucleotide sequence (C), for diagnosing the onset or the risk of the onset of Charcot-Marie-Tooth disease.

Specifically, the CMT may be CMT2.

The term "agent capable of specifically detecting a protein" means a substance that can be used to specifically identify or detect the protein in a sample of an individual.

The agent capable of specifically detecting the protein can be a substance capable of specifically detecting the p.Y118H mutation site.

The term "agent capable of specifically detecting the p.Y118H mutation site of a protein" means a substance that can be used to specifically identify or detect the p.Y118H mutation site of the protein in a sample of an individual .

The agent capable of specifically detecting the protein may be a specific compound or a synthetic substance that targets a HSPB3 protein variant, for example, a mutation site of p.Y118H of the HSPB3 variant, and more specifically, to a HSPB3 protein variant (Y), which is located at position 118 of the protein of SEQ ID NO: 1, is an HSPB3 protein variant in which H is replaced by histidine (H), and more specifically, , But the type thereof is not necessarily limited thereto.

The term "antibody ", as it is known in the art, refers to a specific protein molecule directed against an antigenic site. An antibody specific for the HSPB3 protein variant can be obtained by cloning a gene encoding a HSPB3 protein variant into an expression vector according to a conventional method to obtain an HSPB3 protein variant encoded by the gene, ≪ / RTI > Also included are partial peptides that may be made from the HSPB3 protein variants, and the partial peptides may include at least 7 amino acids, preferably 9 amino acids, and more preferably 12 amino acids. The form of the antibody is not particularly limited, and any polyclonal antibody, monoclonal antibody or antigen-binding property thereof may be included in the antibody and all the immunoglobulin antibodies may be included. Further, the antibody may also include a special antibody such as a humanized antibody.

A polyclonal antibody can be produced by methods well known in the art for injecting an HSPB3 protein variant antigen described above into an animal and obtaining blood from an animal to obtain an antibody-containing serum. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, dogs, and the like.

Monoclonal antibodies can be prepared using the hybridoma method (Kohler and Milstein, European Jounal of Immunology 6: 511-519, 1976) or the phage antibody library (Clackson et al, Nature 352: 624- 628, 1991; Marks et al., J Mol Biol 222 (58): 1-597, 1991). The antibody prepared by the above method can be separated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The antibody capable of detecting the HSPB3 protein variant may comprise a complete form having two full-length light chains and two full-length heavy chains as well as a functional fragment of the antibody molecule. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

The term "agent capable of specifically detecting a polynucleotide" means a substance that can be used to specifically identify or detect the polynucleotide in a sample of an individual.

The agent capable of specifically detecting the polynucleotide can be a substance capable of specifically detecting the c.352T> C mutation site.

The term "agent capable of specifically detecting the c.352T> C mutation site of a polynucleotide" refers to an agent that can specifically detect or detect the c.352T> C mutation site of the polynucleotide in a sample of an individual Means a substance that can be absorbed.

The agent capable of specifically detecting the polynucleotide may be a substance that specifically binds to the HSPB3 gene mutant, specifically, a substance that specifically binds to the c.352T> C mutation site of the HSPB3 gene mutant , more specifically, may be a c.352T> polynucleotide specifically binding to C mutation region of the mutant gene HSPB3, more specifically, to specifically bind to c.352T> C mutation region of the mutant gene HSPB3 primers, probes Or antisense nucleic acid, but the type thereof is not necessarily limited thereto.

The agent capable of specifically detecting the polynucleotide is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or a complementary polynucleotide thereof, wherein the polynucleotide comprises a mutation site of the polynucleotide and comprises 10 or more consecutive Lt; / RTI > polynucleotide. The mutated site may be the 352nd nucleotide from the 5'end of SEQ ID NO: 2.

The agent capable of specifically detecting the polynucleotide indicates a single nucleotide variation at the mutation site. Thus, when one single-stranded polynucleotide is associated with the risk of developing Sharko-Maritus disease, the polynucleotide complementary to the single-stranded polynucleotide is naturally associated with the risk of developing Charko-Mariotys disease Can be judged. Thus, compositions according to one aspect may comprise single-stranded polynucleotides and polynucleotides having a sequence complementary thereto, which are associated with the risk of developing a Charcot-Marie-Tooth disease with one particular sequence. For example, the polynucleotide of SEQ ID NO: 2 is "T or C" for the 352nd nucleotide. In this case, the agent capable of specifically detecting the polynucleotide comprises 10 or more consecutive nucleotides selected from the polynucleotide of SEQ ID NO: 2, including the 352nd "T or C" nucleotide, Stranded < / RTI > polynucleotide with "A or G" That is, the agent capable of specifically detecting the polynucleotide can detect the 352nd nucleotide from the 5'end of the SEQ ID NO: 1, which is a mutation site in the polynucleotide of SEQ ID NO: 2. Or a preparation capable of specifically detecting the polynucleotide can detect the c. 352T > C mutation in the polynucleotide of SEQ ID NO: 2.

The primer, probe, or antisense nucleic acid specifically binds to a polynucleotide consisting of a nucleotide sequence in which the thymine (T) at position 352 of the nucleotide sequence of SEQ ID NO: 2 is replaced with a cytosine (C) And may not be specific binding.

The primer, probe or antisense nucleic acid may be prepared by a conventional method using HSPB3 Can be designed based on the nucleotide sequence of the gene variant.

The term "primer" refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a complementary template and serving as a starting point for template strand copying. Lt; RTI ID = 0.0 > 50 < / RTI > Primers are usually synthesized but can also be used in naturally occurring nucleic acids. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, but is sufficiently complementary that it can hybridize with the template. Primers are designed to initiate DNA synthesis in the presence of a polymerization reaction (i. E., A reagent for DNA polymerase or reverse transcriptase and four different nucleoside triphosphates) at the appropriate buffer solution and temperature The sense of the nucleotide sequence of the HSPB3 gene mutant and the antisense primer can be used to amplify the PCR reaction , the sense and the antisense primer, Amplification can be performed to diagnose CMT.

The term "probe" means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or a few hundred nucleotides that can specifically bind to mRNA, and is labeled to confirm the presence or absence of a specific nucleic acid . The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art. Thus, hybridization can be performed using a probe complementary to the nucleotide sequence of the HSPB3 gene mutant, and CMT can be diagnosed through hybridization.

The primers or probes can be chemically synthesized using the phospharamidite solid support method, or other well-known methods. Such nucleic acid sequences may incorporate additional features that do not alter the basic properties. Examples of additional features that may be incorporated include, but are not limited to, methylation, capping, substitution of one or more nucleic acids with homologues, and modifications between nucleic acids.

The term "antisense nucleic acid" refers to complementary with the sequence I molecules of the nucleic acid base to form a HSPB3 gene mutant and dimer on HSPB3 gene variants that are targeted, and may be used to detect the HSPB3 gene variants.

The antisense nucleic acid may be the polynucleotide or a fragment thereof, or complementary thereto. More preferably 10 to 1000, more particularly 10 to 500, even more specifically 15 to 500, even more specifically 15 to 300, more specifically 10 or more, more specifically 10 to 1000, More specifically from 15 to 200 nucleotides, more particularly from 15 to 150 nucleotides, but it is possible to choose an appropriate length to increase the detection specificity.

Another aspect provides a kit for diagnosing the onset or the risk of the onset of CMT comprising said composition.

The kit comprises a protein consisting of the amino acid sequence in which the tyrosine (Y) at position 118 in the amino acid sequence of SEQ ID NO: 1 is replaced with histidine (H) or the protein consisting of the amino acid sequence at position 352 in the nucleotide sequence of SEQ ID NO: 2 ) Is specifically detected as a polynucleotide consisting of a nucleotide sequence substituted with a cytosine (C), thereby diagnosing whether or not CMT is present or the risk of the onset thereof.

Specifically, the kit comprises a p.Y118H mutation site of the protein consisting of the amino acid sequence in which the tyrosine (Y) at position 118 in the amino acid sequence of SEQ ID NO: 1 is replaced with histidine (H), or the nucleotide sequence of SEQ ID NO: The c.352T> C mutation site of a polynucleotide consisting of a nucleotide sequence in which the thymine (T) at position 352 in the sequence is substituted with a cytosine (C) is specifically detected to diagnose the onset or the risk of CMT .

The kit specifically includes an antibody that specifically recognizes a protein consisting of an amino acid sequence in which tyrosine (Y) at position 118 in the amino acid sequence of SEQ ID NO: 1 is substituted with histidine (H), or an antibody that specifically recognizes a protein at position 352 in the nucleotide sequence of SEQ ID NO: A probe or an antisense nucleic acid capable of specifically detecting a polynucleotide consisting of a nucleotide sequence in which the thymine (T) of the polynucleotide is replaced with a cytosine (C), and further comprising one or more other components Compositions, solutions, or devices.

Specifically, the kit may be an RT-PCR kit, a microarray chip kit, or a protein chip kit.

The RT-PCR kit was designed to specifically detect the c.352T> C mutation site of a polynucleotide consisting of a nucleotide sequence in which the thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 was substituted with cytosine (C) Lt; RTI ID = 0.0 > RT-PCR. ≪ / RTI > In addition to the respective primer pairs specific for the c.352T> C mutation site of the polynucleotide consisting of the nucleotide sequence in which the thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 was substituted with cytosine (C) DNase, RNase inhibitor, DEPC-water, sterilized water, etc., such as a test tube or other appropriate container, reaction buffer, deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase .

The microarray chip may comprise a DNA or RNA polynucleotide probe. The microarray comprises a probe specific for a c.352T> C mutation site of a polynucleotide consisting of a nucleotide sequence in which a thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 is substituted with a cytosine (C) And may include the configuration of a conventional microarray. The microarray detects the presence of a c.352T> C mutation site of a polynucleotide consisting of a nucleotide sequence in which the thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 is substituted with cytosine (C) And can provide useful information to diagnose the likelihood.

A probe for a c.352T> C mutation site of a polynucleotide consisting of a nucleotide sequence in which the thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 was substituted with a cytosine (C) was immobilized on a substrate to prepare a microarray Are well known in the art. For example, a DNA microarray can be prepared by a micropipetting method using a piezo electric method or a method using a spotter in the form of a pin, > Probes for C mutation sites can be immobilized on a substrate. The substrate of the microarray may be coated with an activating group selected from the group consisting of amino-silane, poly-L-lysine and aldehyde, but is not limited thereto . The substrate may be selected from the group consisting of slide glass, plastic, metal, silicon, nylon film, and nitrocellulose membrane, but is not limited thereto.

In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. The detection can be accomplished by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent material, such as Cy3 and Cy5, and then hybridizing on the microarray and detecting a signal The hybridization result can be detected.

The protein chip kit can measure the expression level of a protein consisting of the amino acid sequence in which the tyrosine (Y) at position 118 in the amino acid sequence of SEQ ID NO: 1 is replaced with histidine (H). The protein chip kit may comprise a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, or the like for immunological detection of the antibody. As the substrate, a nitrocellulose membrane, a 96-well plate synthesized with a polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, a slide glass made of glass, or the like can be used as the substrate, and a peroxidase ), Alkaline phosphatase, and the like can be used. As the fluorescent material, FITC, RITC and the like can be used. As the chromogenic substrate, ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid)), OPD (Tetramethylbenzidine), and the like can be used.

Another aspect relates to a method for detecting CMT or providing information for predicting the risk of onset, wherein in a biological sample isolated from an individual, tyrosine (Y) at position 118 in the amino acid sequence of SEQ ID NO: 1 is replaced with histidine Or a polynucleotide consisting of a nucleotide sequence in which a thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 is substituted with a cytosine (C).

The term "individual" refers to an individual who seeks to determine whether or not CMT has developed or is likely to develop or predict the risk of developing CMT. The subject may be a vertebrate animal, specifically a mammal, an amphibian, a reptile, a bird, or the like, more specifically a mammal, for example a human ( Homo sapiens ). The human being may be Korean.

The term "biological sample" may include, but is not limited to, tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine samples separated from the subject. Methods for obtaining biological samples from an individual are known in the art. In the case of a protein sample, the separated tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine can be used directly or proteins dissolved in the lysis buffer can be used. The protein sample includes not only purely separated proteins, but also cell lysates containing crude proteins, for example, proteins. In the case of a nucleic acid sample, a nucleic acid amplified with a specific region can be used by directly separating the nucleic acid from tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine or by a nucleic acid amplification method such as PCR. The nucleic acid may be DNA or RNA. The DNA may be genomic DNA, cDNA, or recombinant DNA. The RNA may be mRNA. The nucleic acid sample includes not only a nucleic acid that is purely isolated, but also a cell lysate containing a crude nucleic acid, for example, a nucleic acid.

The protein may be one that directly detects and detects the amino acid residue at the amino acid mutation site. Methods for determining amino acids are known in the art. For example, a protein sequence analyzer can be used to directly determine the amino acid residue at the amino acid mutation site.

The protein may be detected using an agent capable of specifically detecting the protein. Specifically, the agent capable of specifically detecting the protein may be a preparation capable of specifically detecting the p.Y118H mutation site. The agent capable of specifically detecting the protein may be, but is not limited to, an antibody. The amino acid at the amino acid mutation site can be determined by measuring the binding between the antigen and the antibody.

Methods for detecting the protein include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), Protein Chip, and the like are available. However, But is not limited thereto.

The polynucleotide may be one that directly detects and detects the nucleotide at the nucleotide mutation site. Methods for determining nucleotides are known in the art. For example, the nucleotide at the nucleotide mutation site can be directly determined using a nucleotide sequence analyzer. The nucleotide of the mutation site can be directly determined by a nucleotide sequencing method of a known nucleic acid. Methods for nucleotide sequencing can include ginger (or dideoxy) sequencing methods, next generation sequencing, next generation sequencing, or germ-gilbert (chemical cleavage) methods.

The polynucleotide may be detected using an agent capable of specifically detecting the polynucleotide. Specifically, the agent capable of specifically detecting the polynucleotide may be one capable of specifically detecting the c.352T> C mutation site. The agent capable of specifically detecting the c.352T> C mutation site of the polynucleotide may be, but is not limited to, a primer, a probe, or an antisense nucleic acid. For example, a nucleotide at a mutation site can be determined by hybridizing a probe containing a sequence at a mutation site with a polynucleotide of interest, and analyzing the result of hybridization. The degree of hybridization can be confirmed, for example, by marking a detectable label on the target nucleic acid, detecting the hybridized target nucleic acid, or confirming it by an electrical method or the like. In addition, a single base primer extension (SBE) method can be used.

Methods for detecting the polynucleotide include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RNase protection) assay: RPA), Northern blotting, DNA chip, and the like.

In the above method, a biological sample separated from an individual may include a p.Y118H mutation site of a protein consisting of the amino acid sequence in which the tyrosine (Y) at position 118 in the amino acid sequence of SEQ ID NO: 1 is replaced with histidine (H) The presence of the c.352T> C mutation site of a polynucleotide consisting of a nucleotide sequence in which the thymine (T) at position 352 in the nucleotide sequence of the nucleotide sequence of the polynucleotide of the present invention is replaced by a cytosine (C) .

The "risk of onset of Charcot-Marie-Tooth disease" may be a relative risk of the risk of Charcot-Marie-Tooth. For example, the risk may be indicative of whether the probability of onset is increased relative to a healthy normal population or a group having a reference genomic sequence. In addition, the risk may be that an individual with a particular allele or genotype has an increased likelihood of developing a Charcot-Marie-Tooth disease as compared to an individual having another specific allele or genotype.

The HSPB3 protein mutant or HSPB3 mutant according to one aspect can be used as a marker for diagnosing Sharko- Mariotus disease type 2. The diagnosis using the HSPB3 gene mutant enables accurate early diagnosis of Charcot-Marie-Tooth disease through genetic testing, thereby maximizing the therapeutic effect by applying the recently developed treatment method early.

Figure 1 is a pedigree diagram of a family (c. 352T > C variant) family that includes a CMT2 patient with a HSPB3 mutation. The genotype of the HSPB3 mutation site is shown at the bottom of each member. White figure is normal, black figure is CMT2 patient.
FIG. 2 is a nucleotide sequence chromatogram containing the HSPB3 gene mutation site in the control (normal human) and CMT2 patients.
FIG. 3 shows the result of amino acid sequence analysis showing that the position (p.Tyr118His) of the HSPB3 mutation of the CMT2 patient is well preserved in the species.
Fig. 4 shows the results of confirming conformational changes of p.Tyr118His protein variant.
Figure 5 is an MRI taken from a patient with the p.Tyr118His mutation.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One. CMT and  Identification of associated gene and protein mutations

1. Selection of patients

This study was conducted on a family with autosomal dominant axon CMT2 neuropathy. There were a total of 6 family members, 2 of them were onset, and 4 were not onset. Control subjects were 500 normal subjects, and HSPB3 Genotype of the gene was examined. All participants agreed in writing in accordance with the approved protocol (SMC, 2014-08-057-002) from the Review Committee of Samsung Hospital, Sungkyunkwan University.

2. Identification of total exome sequencing and CMT2 gene

The CMT2 gene was identified in blood samples from CMT2 patients.

Specifically, peripheral blood was obtained from the family members and genomic DNA was extracted using a QIAamp blood DNA purification kit (Qiagen, Hilden, Germany). Hexaplex microsatellite polymerase chain reaction (PCR) and real-time PCR were used to confirm the presence of 17p12 ( PMP22 ) redundancy in patient samples. The genotypes were analyzed using an automated sequencer ABI3130XL and Genemapper software (Life Technologies, Foster City, Calif., USA).

The entire exomes were captured using a SureSelect Human All Exon 50M kit (Agilent Technologies, Santa Clara, Calif., USA), and then the standard pair-ends were obtained using a HiSeq2000 genome analyzer (Illumina, San Diego, CA, USA) lt; RTI ID = 0.0 > paired-end < / RTI > mode. Whole exome sequencing (WES) was performed in Macrogen (Seoul, Korea). UCSC assembly hg19 was used as a reference sequence. The small nucleotide variant (SNV) was extracted using the GATK program and the mutation was annotated using the ANNOVAR program. Sequences were extracted only when the read depth was 5 or more. Annotated SNVs were compared to those registered at dbSNP150 (http://www.ncbi.nlm.nih.gov).

(Such as missense, nonsense, exonic indel, stop gain, and splicing site) that are functionally important can be removed from the data first Respectively. Subsequently, a new or rare mutation (minor allele frequency (MAF) of less than 0.01) was generated by the Genome Project (http://www.1000genomes.org/), the Exome Sequencing Project (ESP ) (http://evs.gs.washington.edu/EVS/), the Exome Aggregation Consortium (ExAC) (http://exac.broadinstitute.org/) database, and the Korean Reference Genome Genome: KRGDB) (http://152.99.75.168/KRGDB/). The mutations obtained from WES were confirmed by the Sanger sequencing method. In order to determine the effect of the mutations on the protein, in silico (i n silico) analyze MUpro (http://www.ics.uci.edu/~baldig/mutation), PolyPhen-2 (http: // genetics. bwh.harvard.edu/pph2), SIFT (http://sift.jcvi.org) and PROVEAN (http://provean.jcvi.org). The genomic evolutionary rate profiling (GERP) score was determined by the GERP ++ program (http://mendel.stanford.edu/SidowLab/downloads/gerp/index.html).

As a result of WES analysis, the foot terminal (IV-2) is a novel heterozygous mutation, HSPB3 Showed c.352T> C mutation in the gene. FIG. 2 is a nucleotide sequence chromatogram containing the HSPB3 gene mutation site in the control (normal human) and CMT2 patients. The c.352T> C mutation was predicted to induce the p.Tyr118His mutation. The c.352T> C mutation occurred in all family members and was examined by sequencing. The patient's father and son had the c.352T> C variant. However, other uninjured families did not have these mutations. This showed a separation of mutations in the affected individuals, except for the patient's 5-year-old son. Because the 5-year-old son did not reach the age of onset, the mutation is not expected to develop into the CMT2 phenotype. The identified mutations were not identified in some global human variation databases, including 500 normal controls and 1000 genome projects, dbSNP147, ESP, and ExAC. FIG. 3 shows the result of amino acid sequence analysis showing that the position (p.Tyr118His) of the HSPB3 mutation of the CMT2 patient is well preserved in the species. As shown in Figure 3, the mutation site was located in the well-conserved alpha-cristalline domain between vertebrate species. Vertebrates human (Homo spiens), mice (Mus musculus), cow (Bos taurus), wolf (Canis lupus familiaris), chicken (Gallus gallus ), African clawed frog ( Xenopus laevis ), and zebrafish ( danio rerio ). As shown in Table 1, for these mutations, the GERP score was very high, 5.71, and some of the silico prediction programs (MUpro, PolyPhen-2, SIFT, and PROVEAN) predicted the mutation to be pathogenic. The structure of the p. Tyr118His mutant protein was confirmed using the I-TASSER program. Fig. 4 shows the results of confirming conformational changes of p.Tyr118His protein variant. As shown in Table 2, from the target sequence analysis results, some rare nonsynonymous mutations in the CMT- or CMT-related genes were confirmed from the target sequence analysis data of the initiator. All mutations were expected to be polymorphic or rare individual mutations and were not co-segregated with the affected family members.

transition Korean ^b dbSNP150 ^c 1000G ^d ESP ^e ExAC ^f In silico analysis ^g GERP ^h Nucleotides ^a amino acid PolyPhen-2 MUpro SIFT PROVEAN c.352T> C p.Y118H No No No No No 1 ^* -0.846 ^* 0 ^* -4.967 ^* 3.60

a. The nucleotide accession number is NM_006308.2.

b. From the total exon sequence data, the Korean healthy controls (n = 500)

c. SNP access number of dbSNP150 (http://www.ncbi.nlm.nih.gov)

d. Variant allelic frequency of the 1000 genome (1000G) database (http://www.1000genomes.org/)

e. Frequency of mutation alleles in the exon sequencing project (ESP) database (http://evs.gs.washington.edu).

f. Frequency of mutation alleles in the exome set consortium (ExAC) database (http://exac.broadinstitute.org).

g. In silico analysis: * indicates anticipated pathogenic.

h. GERP: Genome evolution rate profiling score (http://mendel.stanford.edu/SidowLab/downloads/gerp/index.html).

Gene ^a transition State ^b dbSNP
Registration Number Allele frequency ^c Nucleotides amino acid 1000G ESP ExAC KRGDB PLEKHG5 c.260T> C p.I87T Het rs117505788 0.008 0.0002 0.004 0.0297 DST c.A9516C p.L3172F Het rs781167503 NR NR 0.000017 0.000804 WNK1 c.2220_2221insC p.L740fs Hom rs397768556 NR NR NR 0.5579 SBF1 c.C5311T p.R1771C Het rs138127298 0.001 0.0002 0.0003 0.0048

a. The nucleotide accession numbers are PLEKHG5: NM_020631.4, DST: NM_001723.5, WNK1: NM_213655.4, and SBF1: NM_002972.2.

b. Het: heterozygous, and Hom: homozygous.

c. 1000G, ESP, ExAC, Same as above, KRGDB: Korean Reference Genome database.

3. CMT2  Clinical Signs and Electrophysiological Evaluation of Patients

Experiments were conducted to confirm clinical signs of CMT patients.

Specifically, we identified motor and sensory disturbances, deep tendon reflexes, and atrophy in CMT2 patients. The age at onset was determined by asking the patient the age of the patient who first presented with CMT symptoms. The strength of the flexor and extensor was manually measured on a medical research council (MRC) scale. The severity of the disease was assessed using a functional disability scale of 9 (FDS). The motor and sensory conduction velocities of the median nerve, ulnar nerve, peroneal nerve, and calf nerve were measured. The motor nerve conduction velocity (MNCV) of the median nerve and the ulnar nerve was measured by stimulation at the elbow or wrist, while the composite of the abductor pollicis brevis and the adductor digiti quintion The compound muscle action potential (CMAP) was recorded. In the same way, the CMAP of the extensor digitorum brevis was recorded, while measuring the MNCV of the peroneal nerve by stimulation at the knee and ankle. CAMP amplitude was measured from baseline to negative peak value. Sensory nerve conduction velocity (SNCV) was obtained from the median nerve and ulnar nerve by orthodromic scores for the finger-wrist region, and also for the calf nerves. Sensory nerve action potential (SNAP) amplitude was also measured.

Clinical characteristics and electrophysiological analysis results of CMT2 patients are shown in Table 3 below. Figure 1 is a pedigree diagram of a family (c. 352T > C variant) family that includes a CMT2 patient with a HSPB3 mutation. The genotype of the HSPB3 mutation site is shown at the bottom of each member. White figure is normal, black figure is CMT2 patient. The originator was a 29-year-old woman (Fig. 1, IV-2), showing progressive distal leg weakening. At 21 years of age, frequent falls accompanied by gait disturbance and instability in climbing stairs were observed. At the age of 29, walking disorders progressed gradually, and ankle assistance began to be used. Neurological examination showed distal muscle weakness and atrophy mainly in the lower limb, regardless of the proximal muscle. Muscle atrophy was more prominent in the distal limb than in the distal limb. Pals cavus, extensive gait, and atrophy of foot and calf muscles were observed, but scoliosis was not observed. The great toe plantar flexors were graded 0 to 1, while the great toe dorsiflexor was grade 4, which was consistent with lower limb MRI findings, as shown in FIG. 5e. Ankle dorsiflexors and evertor were grade 4 and ankle plantar flexors and invertors were grade 4 or higher. The patient had a steppage gait and was unable to walk with the heel and toe. In this patient, length-dependent sensory loss was observed, and vibration and position sensation were severely impaired than pain and temperature sensation. There was anti-reflex (Areflexia) in the upper and lower limbs, but no pathological reflexes were found. The patient had a moderate level of physical disability (CMT neuropathy score: CMTNS) and a grade 3 FDS.

The patient showed a delayed exercise latency with reduced CMAP in the peroneal nerve. There was almost no SNAP in the calf nerves, and SNAP and SNCV decreased in both the median and ulnar nerves. Needle electrode examination showed abnormal and spontaneous activity of hands and distal leg muscles with chronic denervation symptoms in all lower extremities and hand muscles. Visual evoked potentials and auditory evoked potentials were normal.

In order to confirm the family history of the patient, a 57-year old father (FIG. 1, III-1) and a 84-year old grandmother (FIG. 1, II-3) were examined. The father of the patient showed disturbance of the distal upper limb and lower limbs, accompanied by atrophy, and showed a bony, and walked in the gait. Vibration and position sensation were more severely impaired than pain and temperature sensations. The patient 's father showed no antiglare in the upper limb and lower limbs, but no pathological reflex was found. Patient 's grandmother also showed distal dominant muscle weakness and was dependent on wheelchair after age 73. The patient's son (Fig. 1, V-1, 3 years old) appeared to be onset but slightly younger to show symptoms. Patients' mother (Figure 1, III-2, 56 years old) showed no neuromuscular symptoms and was healthy.

Phenotype CMT2 transition Change nucleotide c.352T> C Amino acid change p.Y118H domain Alpha-cristalline domain
(alpha-crystalline domain) heredity Autosomal dominant gender female Age of test (age) 29 Age at onset (age) 21 Symptoms at onset Gait disorder Muscle weakness Gradual progression circle distal dominance
(slowly progressive distal dominant) Muscle atrophy Upper limb Lower limb Sensory loss Yes (vibration> pin prick) Pes cavus Yes Steppage gait Yes Knee / ankle jerks - / - Motor nerve Median motor nerve TL (ms) 3.1 CMAP (mV) 25.1 MNCV (m / s) 48.1 Ulnar motor nerve TL (ms) 2.5 CMAP (mV) 12.8 MNCV (m / s) 50.6 Peroneal nerve TL (ms) 6.9 CMAP (mV) 1.5 MNCV (m / s) 29.9 Sensory nerve NCS Median sensory nerve SNAP (μV) 5.1 SNCV (m / s) 32.2 Ulnar sensory nerve SNAP (μV) 6.4 SNCV (m / s) 33.5 Calf nerves SNAP (μV) A SNCV (m / s) A

A: Absent, CMAP: compound muscle action potential, CMTNS: Charcot-Marie-Tooth neuropathy score, FDS: functional disability scale, MNCV: Sensory nerve conduction velocity, motor nerve conduction velocity, SNAP: sensory nerve action potential, SNCV: sensory nerve conduction velocity,

Normal NCV (m / s): Exercise median nerve ≥ 50.5, ulnar nerve ≥ 51.1, peroneal nerve ≥ 41.3, sensory nerve ≥ 39.3, ulnar nerve ≥ 37.5, calf nerve ≥ 32.1.

Normal amplitude (mV): Exercise median nerve ≥ 6, ulnar nerve ≥ 8, peroneal nerve ≥ 1.6, sensory median nerve (μV) ≥ 8.8, ulnar nerve ≥ 7.9, calf nerve ≥ 6.0.

3. CMT2  Identification of fatty replacement in patient's leg muscles

Magnetic resonance imaging (MRI) of the thigh and calf of CMT2 patients was performed to confirm the finding that the muscles of the CMT2 patients were replaced with fat.

Specifically, MRI was obtained using a 1.5-T system (Siemens Vision, Siemens, Germany) equipped with a phase-array multi-coil. The patient 's thighs and calves were subjected to MRI. The leg MRI is an axial view (field of view: FOV, 24 to 32 cm; Section thickness, 10 mm; 0.5 to 1.0 mm] and coronal plane (FOV, 38 to 40 cm; section thickness, 4 to 5 mm; section interval, 0.5 to 1.0 mm) spin-echo: SE) (TR / TE 570-650 / 14-20, 512 matrix), T2-weighted SE (TR / TE 2800-4000 / 96-99, 512 matrix) and fat- A protocol of T2-weighted SE (TR / TE 3090-4900 / 85-99, 512 matrix) was used.

FIG. 5 is an MRI image of a patient's leg having a p.Tyr118His mutation. As shown in FIG. 5, T1-weighted MRI showed a significant degree of fat replacement and muscle atrophy in the lower limbs muscle compared with the thigh muscle, consistent with the theory of length dependent neuropathy. When the patient was 29 years old, T1-weighted MRI showed severe muscle atrophy in the calf muscle and dominance in the distal portion. Semitendinous muscle, adductor muscle, vastus medialis, and intermedius muscle are relatively less damaging in the hip and thigh, as shown in Figures 5a, 5c and 5d . 5e and 5b, in the calf, the soleus muscle (white arrow), the gastrocnemius muscle and the peronei muscle showed severe disturbances and the anterior muscle of the tibia (arrow) had a slight fat Infiltration.

<110> Samsung Life Public Welfare Foundation KONGJU NATIONAL UNIVERSITY INDUSTRY-UNIVERSITY COOPERATION FOUNDATION <120> Marker for diagnosing Charcot-Marie-Tooth disease and use thereof <130> PN117904 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 150 <212> PRT <213> Homo sapiens <400> 1 Met Ala Lys Ile Ile Leu Arg His Leu Ile Glu Ile Pro Val Arg Tyr   1 5 10 15 Glu Glu Glu Phe Glu Ala Arg Gly Leu Glu Asp Cys Arg Leu Asp His              20 25 30 Ala Leu Tyr Ala Leu Pro Gly Pro Thr Ile Val Asp Leu Arg Lys Thr          35 40 45 Arg Ala Ala Gln Ser Pro Ala Ala Glu Thr Pro Pro      50 55 60 Arg Glu Gly Lys Ser His Phe Gln Ile Leu Leu Asp Val Val Gln Phe  65 70 75 80 Leu Pro Glu Asp Ile Ile Gln Thr Phe Glu Gly Trp Leu Leu Ile                  85 90 95 Lys Ala Gln His Gly Thr Arg Met Asp Gly His Gly Phe Ile Ser Arg             100 105 110 Ser Phe Thr Arg Gln Tyr Lys Leu Pro Asp Gly Val Glu Ile Lys Asp         115 120 125 Leu Ser Ala Val Leu Cys His Asp Gly Ile Leu Val Val Glu Val Lys     130 135 140 Asp Pro Val Gly Thr Lys 145 150 <210> 2 <211> 453 <212> DNA <213> Homo sapiens <400> 2 atggcaaaaa tcattttgag gcacctcata gagattccag tgcgttacca ggaagagttt 60 gaagctcgag gtctagaaga ctgcaggctg gatcatgctt tatatgcact gcctgggcca 120 accatcgtgg acctgaggaa aaccagggca gcgcagtctc ctccagtgga ctcagcggca 180 gagacgccac cccgagaagg caaatcccac tttcagatcc tgctggacgt ggtccagttc 240 ctccctgaag acatcatcat tcagaccttc gaaggctggc tgctgataaa agcacaacac 300 ggaaccagaa tggatgagca cggttttatc tcaagaagct tcacccgaca gtacaaacta 360 ccagatggtg tggaaatcaa agatttgtct gcagtcctct gtcatgatgg aattttggtg 420 gtggaagtaa aggatccagt tgggactaag tga 453

Claims (22)

A heat shock protein family member 3 (HSPB3) protein variant consisting of the amino acid sequence in which the tyrosine (Y) at position 118 in the amino acid sequence of SEQ ID NO: 1 is replaced with histidine (H). A polynucleotide encoding a HSPB3 protein variant according to claim 1. The polynucleotide according to claim 2, wherein the polynucleotide comprises a nucleotide sequence in which the thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 is replaced with a cytosine (C). A protein consisting of an amino acid sequence in which the tyrosine (Y) at position 118 in the amino acid sequence of SEQ ID NO: 1 is substituted with histidine (H)
A preparation capable of specifically detecting a polynucleotide consisting of a nucleotide sequence in which a thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 has been substituted with a cytosine (C)
A composition for diagnosing the risk of, or risk of developing, Charcot-Marie-Tooth disease (CMT). The composition according to claim 4, wherein the agent capable of specifically detecting the protein is an agent capable of specifically detecting the p.Y118H mutation site. The composition according to claim 4, wherein the agent capable of specifically detecting the protein is an antibody. The composition according to claim 4, wherein the agent capable of specifically detecting the polynucleotide is an agent capable of specifically detecting c.352T> C mutation site. The composition according to claim 4, wherein the agent capable of specifically detecting the polynucleotide is a primer, a probe, or an antisense nucleic acid. 9. The composition of claim 8, wherein the antisense nucleic acid is complementary to the polynucleotide or fragment thereof. 10. The composition of claim 9, wherein the fragment has at least 10 nucleotides. 5. The composition of claim 4, wherein the CMT is Charcot-Marie-Tooth disease type 2 (CMT2). A kit for diagnosing the onset or the risk of the onset of CMT comprising the composition of any one of claims 4 to 11. 13. The kit of claim 12, wherein the kit is an RT-PCR kit, a microarray chip kit, or a protein chip kit. In order to provide information for diagnosing CMT or for predicting the risk of onset, in a biological sample separated from an individual,
A protein consisting of an amino acid sequence in which the tyrosine (Y) at position 118 in the amino acid sequence of SEQ ID NO: 1 is substituted with histidine (H)
A method for detecting a polynucleotide comprising a nucleotide sequence in which a thymine (T) at position 352 in the nucleotide sequence of SEQ ID NO: 2 is substituted with a cytosine (C). 15. The method according to claim 14, wherein the protein is detected using an agent capable of specifically detecting the protein. 16. The method according to claim 15, wherein the agent capable of specifically detecting the protein is an agent capable of specifically detecting the p.Y118H mutation site. 16. The method of claim 15, wherein the agent capable of specifically detecting the protein is an antibody. 15. The method of claim 14, wherein the assay for detecting the protein is selected from the group consisting of Western blotting, ELISA, radioimmunoassay, radioimmunoprecipitation, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, Complement fixation assay, FACS or protein chip. 15. The method according to claim 14, wherein the polynucleotide is detected using a preparation capable of specifically detecting the c.352T> C mutation site of the polynucleotide. The method according to claim 19, wherein the agent capable of specifically detecting the c.352T> C mutation site of the polynucleotide is a primer, a probe, or an antisense nucleic acid. 15. The method according to claim 14, wherein the assay for detecting the polynucleotide is a reverse transcription polymerase chain reaction, a competitive reverse transcription polymerase chain reaction, a real time reverse transcription polymerase chain reaction, an RNase protection assay (RPA), a Northern blotting or a DNA chip. 15. The method of claim 14, wherein the CMT is Charcot-Marie-Tooth disease type 2 (CMT2).

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