KR102387354B1 - Marker for diagnosing Charcot-Marie-Tooth disease and use thereof - Google Patents

Abstract

Charcot-Marie-Tooth disease (CMT) comprising a glycyl-tRNA synthetase (GARS) protein variant or GARS gene variant, and an agent capable of specifically detecting them A composition or kit for diagnosis, and a method for diagnosing the disease using the same, and the GARS protein mutant or GARS gene mutant according to an aspect may be used as a marker for diagnosing Charcot-Marie-Tooth disease. Diagnosis using the GARS gene mutant enables accurate early diagnosis of Charcot-Marie-Tooth disease through genetic testing, and accordingly, treatment effects that have been recently developed can be applied early to maximize the therapeutic effect.

Description

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

Charcot-Marie-Tooth disease (CMT) comprising a glycyl-tRNA synthetase (GARS) protein variant or GARS gene variant, and an agent capable of specifically detecting them It relates to a composition or kit for diagnosing, and a method for diagnosing the disease using the same.

Charcot-Marie-Tooth disease (CMT) is a disease showing symptoms of distal muscle weakness accompanied by loss of sensory function, also called hereditary motor and sensory neuropathy. Charcot-Marie-Tooth disease is mainly characterized by progressive muscle weakness and atrophy in the peroneal muscles of the lower extremities, and is characterized by areflexia, distal sensory loss, pes cavus, and hearing impairment. (deafness) is also indicated. The onset mainly occurs around the age of 10, but rarely occurs after the age of 30. However, the clinical phenotype and age of onset of Charcot-Marie-Tooth disease are very heterogeneous, and nerve biopsy for confirmation is not easy, making accurate diagnosis and treatment difficult (Berger P., Young P., Suter). U. Neurogenet. 4: 1-15 (2002)).

Recently, with the development of human genome research and bioinformatics, research to isolate the causative genes of genetic diseases and to elucidate molecular biological mechanisms is being actively conducted, but the development of an efficient diagnostic method for Charcot-Marie-Tooth disease is still insufficient. For the customized treatment of Charcot-Marie-Tooth disease, which is genetically very heterogeneous, it is first required to establish an accurate genetic diagnosis method. Accordingly, the present inventors have made intensive research efforts to identify the cause of Charcot-Marie-Tooth disease. As a result, a novel mutated gene that has not been previously reported has been identified, and the mutated gene is usefully used in the diagnosis of Charcot-Marie-Tooth disease. The present invention was completed by confirming that it can be

One aspect provides a glycyl-tRNA synthetase (GARS) protein variant or a polynucleotide encoding the same.

Another aspect provides a composition for diagnosing whether or at risk of developing Charcot-Marie-Tooth disease.

Another aspect provides a kit for diagnosing whether or not the onset of Charcot-Marie-Tooth disease or the risk of the onset, comprising the composition.

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

One aspect provides a GARS protein variant in which proline, which is the 724th amino acid residue in the amino acid sequence of SEQ ID NO: 1, is substituted with histidine.

The GARS protein variant may be an isolated GARS protein variant.

The GARS protein is a glycyl-tRNA synthetase, and may be a protein registered as NCBI Accession number NP_002038.2, for example, a protein consisting of the amino acid sequence shown in SEQ ID NO: 1. In addition, the GARS gene is a gene encoding the GARS protein, and may be a gene registered with NCBI Accession number NM_002047.4, for example, a gene including the nucleotide sequence shown in SEQ ID NO: 2.

As used herein, the term "GARS protein variant" may mean that a mutation has occurred in a portion of the amino acid sequence of the GARS protein represented by SEQ ID NO: 1. Specifically, it may be a protein encoded by a GARS gene mutant in which cytosine (C), which is the 2171th base in the nucleotide sequence of SEQ ID NO: 2, is substituted with adenine (A), and more specifically, the amino acid sequence shown in SEQ ID NO: 1 It may be a GARS protein variant in which proline, which is the 724th amino acid residue, is substituted with histidine.

As used herein, the term " GARS gene variant" may mean that a mutation has occurred in a portion of the nucleotide sequence of the GARS gene represented by SEQ ID NO: 2. Specifically, it may be a polynucleotide including a nucleotide sequence in which cytosine (C), which is the 2171th base in the nucleotide sequence of SEQ ID NO: 2, is substituted with adenine (A).

Another aspect provides a polynucleotide encoding the GARS protein variant.

The polynucleotide encoding the GARS protein variant may be a polynucleotide including a nucleotide sequence in which cytosine (C), which is the 2171th base in the nucleotide sequence of SEQ ID NO: 2, is substituted with adenine (A).

In the amino acid sequence of SEQ ID NO: 1, the GARS protein variant consisting of an amino acid sequence in which proline, the 724th amino acid residue, is substituted with histidine, or cytosine (C), the 2171th base in the nucleotide sequence of SEQ ID NO: 2, is substituted with adenine (A) A GARS gene variant comprising a modified nucleotide sequence may be a diagnostic marker for Charcot-Marie-Tooth disease. Accordingly, the p.P724H mutation site of the GARS protein variant or the c.2171C>A mutation site of the GARS gene variant may be a mutation marker for the diagnosis of Charcot-Marie-Tooth disease.

Another aspect provides a twinkle protein variant in which arginine, which is the 391th amino acid residue in the amino acid sequence of SEQ ID NO: 3, is substituted with histidine, and the 406th amino acid residue, arginine, is terminated without being translated by a stop codon.

Twinkle protein is a mitochondrial protein also called "twinkle mtDNA helicase", and may be a protein registered with NCBI Accession number NP_068602.2, for example, it may be a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 there is. In addition, the TWNK gene is a gene encoding the twinkle protein, and may be mixed with " C10orf2 gene", may be a gene registered with NCBI Accession number NM_021830.5, for example, the nucleotide sequence shown in SEQ ID NO: 4 It may be a gene comprising

As used herein, the term "twinkle protein variant" may mean that a mutation has occurred in a portion of the amino acid sequence of the twinkle protein represented by SEQ ID NO: 3. Specifically, in the nucleotide sequence of SEQ ID NO: 4, guanine (G), the 1172th base, is substituted with adenine (A), and cytosine (C), the 1216th base, is substituted with thymine (T). It may be a protein, and more specifically, in the amino acid sequence shown in SEQ ID NO: 3, arginine, the 391th amino acid residue, is substituted with histidine, and the 406th amino acid residue, arginine, is untranslated and terminated by a stop codon. can

As used herein, the term " TWNK gene variant" may mean that a mutation has occurred in a portion of the nucleotide sequence of the TWNK gene represented by SEQ ID NO: 4. Specifically, in the nucleotide sequence of SEQ ID NO: 4, guanine (G), the 1172th base, is substituted with adenine (A), and cytosine (C), the 1216th base, is substituted with thymine (T) A polynucleotide comprising a nucleotide sequence can be

In the amino acid sequence of SEQ ID NO: 3, arginine, the 391th amino acid residue, is substituted with histidine, and the 406th amino acid residue, arginine, is untranslated by a stop codon. A TWNK gene variant comprising a nucleotide sequence in which guanine (G), the 1172th base in the sequence, is substituted with adenine (A), and cytosine (C), the 1216th base, is substituted with thymine (T) is Charcot-Marie-Tooth disease may be a diagnostic marker of Therefore, the p.R391H and p.R406X mutation sites of the twinkle protein variant or the c.1172G>A and c.1216C>T mutation sites of the TWNK gene variant are mutation markers for the diagnosis of Charcot-Marie-Tooth disease. can

In another aspect, in the amino acid sequence of SEQ ID NO: 5, the 18th amino acid residue threonine is substituted with isoleucine, the 38th amino acid residue valine is substituted with leucine, or the 88th amino acid residue alanine is substituted with valine, or the 124th amino acid A frameshift occurs with the residue lysine as a starting point, so that the number of amino acids up to the last amino acid residue including the lysine becomes 72, or methionine, the 194th amino acid residue, is substituted with arginine, or the 194th amino acid residue, methionine, is Provided are variants of the GJB1 protein in which threonine is substituted or arginine at the 215th amino acid residue is substituted with glutamine.

GJB1 protein is a transmembrane protein known as "Gap junction beta-1 protein (GJB1)" or "connexin 32", registered under NCBI Accession number NP_000157.1 It may be a protein that has been used, for example, it may be a protein consisting of the amino acid sequence shown in SEQ ID NO: 5. In addition, the GJB1 gene is a gene encoding the GJB1 protein, and may be a gene registered with NCBI Accession number NM_000166.6, for example, a gene including the nucleotide sequence represented by SEQ ID NO: 6.

As used herein, the term "GJB1 protein mutant" may mean that a mutation has occurred in a portion of the amino acid sequence of the GJB1 protein represented by SEQ ID NO: 5. Specifically, in the nucleotide sequence of SEQ ID NO: 6, cytosine (C), the 53rd base, is substituted with thymine (T), guanine (G), the 112th base, is substituted with thymine (T), or cytosine ( C) is substituted with thymine (T), two adenines (AA) are inserted between bases 371 and 372, thymine (T) at base 581 is substituted with guanine (G), or base 581 is It may be a protein encoded by a GJB1 gene mutant in which thymine (T) is substituted with cytosine (C), or guanine (G), which is the 644th base, is substituted with adenine (A), more specifically, of SEQ ID NO: 5 In the amino acid sequence, the 18th amino acid residue threonine is substituted with isoleucine, the 38th amino acid residue valine is substituted with leucine, the 88th amino acid residue alanine is substituted with valine, or the 124th amino acid residue lysine is the starting point A frameshift occurs so that the number of amino acids up to the last amino acid residue including the lysine is 72, or methionine at the 194th amino acid residue is substituted with arginine, or methionine at the 194th amino acid residue is substituted with threonine, or 215 It may be a GJB1 protein variant in which the th amino acid residue, arginine, is substituted with glutamine.

As used herein, the term " GJB1 gene mutant" may mean that a mutation has occurred in a portion of the nucleotide sequence of the GJB1 gene represented by SEQ ID NO: 6. Specifically, in the nucleotide sequence of SEQ ID NO: 6, cytosine (C), the 53rd base, is substituted with thymine (T), guanine (G), the 112th base, is substituted with thymine (T), or cytosine ( C) is substituted with thymine (T), two adenines (AA) are inserted between bases 371 and 372, thymine (T) at base 581 is substituted with guanine (G), or base 581 is It may be a polynucleotide including a nucleotide sequence in which thymine (T) is substituted with cytosine (C) or guanine (G), which is the 644th base, is substituted with adenine (A).

In the amino acid sequence of SEQ ID NO: 5, the 18th amino acid residue threonine is substituted with isoleucine, the 38th amino acid residue valine is substituted with leucine, the 88th amino acid residue alanine is substituted with valine, or the 124th amino acid residue is A frameshift occurs with lysine as the starting point, so that the number of amino acids up to the last amino acid residue including the lysine becomes 72, or methionine, the 194th amino acid residue, is substituted with arginine, or the 194th amino acid residue, methionine, is replaced with threonine substituted or GJB1 protein variant consisting of an amino acid sequence in which arginine at position 215 is substituted with glutamine, or cytosine (C) at position 53 in the nucleotide sequence of SEQ ID NO: 6 is substituted with thymine (T), or at position 112 The base guanine (G) is substituted with thymine (T), the 263th base cytosine (C) is substituted with thymine (T), or two adenines (AA) are inserted between the 371st and 372th bases, Thymine (T) at position 581 is substituted with guanine (G), thymine (T) at position 581 is substituted with cytosine (C), or guanine (G) at position 644 is substituted with adenine (A) The GJB1 gene mutant comprising a modified nucleotide sequence may be a diagnostic marker for Charcot-Marie-Tooth disease.

Thus, the p.T18I, p.V38L, p.A88V, p.K124fs*72, p.M194R, p.M194T, or p.R215Q mutation site of the GJB1 protein variant, or c.53C of the GJB1 gene variant> The T, c.112G>T, c.263C>T, c.371_372insAA, c.581T>G, c.581T>C, or c.644G>A mutation site is a mutation for the diagnosis of Charcot-Marie-Tooth disease. It may be a marker.

Another aspect provides a variant of the SCN10A protein in which arginine, which is the 1250th amino acid residue in the amino acid sequence of SEQ ID NO: 7, is substituted with glutamine.

The SCN10A protein is a "sodium voltage-gated channel alpha subunit 10 (SCN10A) protein", and may be a protein registered as NCBI Accession number NP_006505.3, for example as shown in SEQ ID NO: 7 It may be a protein consisting of an amino acid sequence. In addition, the SCN10A gene is a gene encoding the SCN10A protein, and may be a gene registered with NCBI Accession number NM_006514.3, for example, a gene including the nucleotide sequence represented by SEQ ID NO: 8.

As used herein, the term "SCN10A protein variant" may mean that a mutation has occurred in a portion of the amino acid sequence of the SCN10A protein represented by SEQ ID NO: 7. Specifically, it may be a protein encoded by a SCN10A gene mutant in which guanine (G), which is the 3749th base in the nucleotide sequence of SEQ ID NO: 8, is substituted with adenine (A), and more specifically, the amino acid sequence shown in SEQ ID NO: 7 It may be a SCN10A protein variant in which arginine, which is the amino acid residue at position 1250, is substituted with glutamine.

As used herein, the term " SCN10A gene variant" may mean that a mutation has occurred in a portion of the nucleotide sequence of the SCN10A gene represented by SEQ ID NO: 8. Specifically, it may be a polynucleotide including a nucleotide sequence in which guanine (G), which is the 3749th base in the nucleotide sequence of SEQ ID NO: 8, is substituted with adenine (A).

SCN10A protein variant consisting of an amino acid sequence in which arginine, which is the 1250th amino acid residue in the amino acid sequence of SEQ ID NO: 7, is substituted with glutamine, or guanine (G), which is the 3749th base in the nucleotide sequence of SEQ ID NO: 8, is substituted with adenine (A) The SCN10A gene mutant comprising a modified nucleotide sequence may be a diagnostic marker for Charcot-Marie-Tooth disease. Accordingly, the p.R1250Q mutation site of the SCN10A protein mutant or the c.3749G>A mutation site of the SCN10A gene mutant may be a mutation marker for the diagnosis of Charcot-Marie-Tooth disease.

A person skilled in the art will be able to easily identify the position of the mutation, the nucleotide sequence, and the amino acid sequence using the accession numbers of the proteins or genes. The specific sequence corresponding to the number registered in the UCSC genome browser or GenBank may be slightly changed over time. It will be apparent to those skilled in the art that the scope of the present invention also extends to such altered sequences.

As used herein, the term "Charcot-Marie-Tooth disease (CMT)" is a disease showing symptoms of distal muscle weakness accompanied by loss of sensory function, depending on the phenotype or genotype, It can be classified as CMT1, CMT2, CMT4, IntCMT or CMTX. CMT1 refers to a demyelinating neurological disease with an autosomal dominant inheritance among CMT diseases and a slow nerve conduction test speed, and may be CMT1A, CMT1B, CMT1C, CMT1D, CMT1E, CMT1F, or CMT1X. CMT2 is an autosomal dominant or recessive inherited axonal neurological disease. It refers to a neurological disease with a different degree of disability or age group than CMT1, and a normal or slightly decreased nerve conduction speed. CMT2A1, CMT2A2, CMT2B, CMT2C, CMT2D Can be , CMT2E, CMT2F, CMT2G, CMT2H, CMT2J, CMT2K, CMT2L, CMT2M, CMT2N, CMT2O, CMT2P, CMT2Q, CMT2R, CMT2S, CMT2T, CMT2U, CMT2V, or CMT2 with giant axons. CMT4 refers to a peripheral sensorimotor polyneuropathy with autosomal recessive inheritance and autosomal recessive demyelination, CMT4A, CMT4B1, CMT4B2, CMT4B3, CMT4C, CMT4D, CMT4E, CMT4F, CMT4G, CMT4H, CMT4J, CMT4K or CMT4X can be IntCMT refers to a neurological disease showing axonal and/or demyelinating features. CMTX is a peripheral sensorimotor polyneuropathy that is related to the X chromosome, progresses slowly and gradually, and weakens and atrophys extremity muscles related to the feet, legs, and hands. CMTX1, CMTX2, CMTX3, CMTX4, CMTX5 or CMTX6 can

As used herein, the term “diagnosing” refers to ascertaining the presence or characteristics of a pathological condition. Accordingly, the "diagnosis of Charcot-Marie-Tooth disease" may mean confirming whether or not Charcot-Marie-Tooth disease develops or the possibility of the onset or predicting the risk of developing Charcot-Marie-Tooth disease.

As used herein, the term "diagnosis marker" may refer to a substance capable of diagnosing whether or not the Charcot-Marie-Tooth disease is onset or the possibility of the onset of the disease.

Selection and application of significant diagnostic markers can determine the reliability of diagnostic results. A significant diagnostic marker may mean a marker with high reliability because the result obtained by diagnosis is accurate, and thus has high validity and shows consistent results even during repeated measurement.

As the diagnostic marker, a GARS protein variant consisting of an amino acid sequence in which proline, the 724th amino acid residue in the amino acid sequence of SEQ ID NO: 1 is substituted with histidine, or cytosine (C), which is the 2171th base in the nucleotide sequence of SEQ ID NO: 2, is adenine ( A) The GARS gene variant containing the nucleotide sequence substituted with A) is a highly reliable marker that is detected only in patients with Charcot-Marie-Tooth disease and has little probability of being detected in a normal control group.

In addition, as the diagnostic marker, in the amino acid sequence of SEQ ID NO: 3, arginine, the 391th amino acid residue, is substituted with histidine, and the 406th amino acid residue, arginine, is a twinkle protein variant consisting of an amino acid sequence that is terminated without being translated by a stop codon; Or, in the nucleotide sequence of SEQ ID NO: 4, guanine (G), the 1172th base, is substituted with adenine (A), and cytosine (C), the 1216th base, is substituted with thymine (T). TWNK gene variant comprising a nucleotide sequence It is detected only in patients with Charcot-Marie-Tooth disease, and is a highly reliable marker with little probability of being detected in a normal control group.

In addition, as the diagnostic marker, in the amino acid sequence of SEQ ID NO: 5, threonine at the 18th amino acid residue is substituted with isoleucine, valine at the 38th amino acid residue is substituted with leucine, or alanine at the 88th amino acid residue is substituted with valine, or , A frameshift occurs from the 124th amino acid residue, lysine, as a starting point, so that the number of amino acids up to the last amino acid residue including the lysine becomes 72, or methionine, the 194th amino acid residue, is substituted with arginine, or the 194th amino acid GJB1 protein variant consisting of an amino acid sequence in which the residue methionine is substituted with threonine or the amino acid residue 215 is substituted with glutamine, or cytosine (C), the 53rd base in the nucleotide sequence of SEQ ID NO: 6, is thymine (T) , the 112th base guanine (G) is substituted with thymine (T), the 263th base cytosine (C) is substituted with thymine (T), or two adenines ( AA) is inserted, thymine (T) at the 581th base is substituted with guanine (G), thymine (T) at the 581th base is substituted with cytosine (C), or guanine (G) at the 644th base is The GJB1 gene mutant containing a nucleotide sequence substituted with adenine (A) is a highly reliable marker that is detected only in patients with Charcot-Marie-Tooth disease and has little probability of being detected in normal controls.

In addition, as the diagnostic marker, the SCN10A protein variant consisting of an amino acid sequence in which arginine, which is the 1250th amino acid residue in the amino acid sequence of SEQ ID NO: 7, is substituted with glutamine, or guanine (G), which is the base 3749 in the nucleotide sequence of SEQ ID NO: 8 The SCN10A gene mutant comprising a nucleotide sequence substituted with adenine (A) is a highly reliable marker that is detected only in patients with Charcot-Marie-Tooth disease and has little probability of being detected in a normal control group.

Therefore, a diagnosis result based on a result obtained by detecting the presence or absence of the variants is reasonably reliable.

Another aspect is a protein consisting of an amino acid sequence in which proline, the 724th amino acid residue, is substituted with histidine in the amino acid sequence of SEQ ID NO: 1, or cytosine (C), the 2171th base in the nucleotide sequence of SEQ ID NO: 2, is substituted with adenine (A) Provided is a composition for diagnosing the onset or risk of Charcot-Marie-Tooth disease, comprising an agent capable of specifically detecting a polynucleotide comprising a modified nucleotide sequence.

The composition is a protein consisting of an amino acid sequence in which arginine, the 391th amino acid residue in the amino acid sequence of SEQ ID NO: 3, is substituted with histidine, and the 406th amino acid residue, arginine, is terminated without being translated by a stop codon, or the nucleotide of SEQ ID NO: 4 A polynucleotide comprising a nucleotide sequence in which guanine (G), the 1172th base in the sequence, is substituted with adenine (A), and cytosine (C), the 1216th base, is substituted with thymine (T) can be specifically detected It may further include an agent.

The composition comprises an amino acid sequence in which threonine, which is the 18th amino acid residue in the amino acid sequence of SEQ ID NO: 5, is substituted with isoleucine, an amino acid sequence in which valine, which is the 38th amino acid residue, is substituted with leucine, in the amino acid sequence of SEQ ID NO: 5, From the amino acid sequence in which alanine, the 88th amino acid residue in the amino acid sequence, is substituted with valine, and lysine, the 124th amino acid residue in the amino acid sequence of SEQ ID NO: 5, a frameshift occurs to the last amino acid residue including the lysine An amino acid sequence in which the number of amino acids is 72, an amino acid sequence in which methionine, the 194th amino acid residue in the amino acid sequence of SEQ ID NO: 5, is substituted with arginine, an amino acid sequence in which methionine, the 194th amino acid residue, in the amino acid sequence of SEQ ID NO: 5 is substituted with threonine , an amino acid sequence in which arginine, the 215th amino acid residue in the amino acid sequence of SEQ ID NO: 5, is substituted with glutamine, and an amino acid sequence in which arginine, the 1250th amino acid residue, is substituted with glutamine in the amino acid sequence of SEQ ID NO: 7 Group consisting of each protein Any one or more proteins selected from, or a nucleotide sequence in which cytosine (C), the 53rd base in the nucleotide sequence of SEQ ID NO: 6, is substituted with thymine (T), guanine (G), the 112th base in the nucleotide sequence of SEQ ID NO: 6 The nucleotide sequence substituted with this thymine (T), the nucleotide sequence in which cytosine (C), which is the 263th base in the nucleotide sequence of SEQ ID NO: 6, is substituted with thymine (T), the 371th and 372th bases in the nucleotide sequence of SEQ ID NO: 6 A nucleotide sequence with two adenine (AA) inserted between them, a nucleotide sequence in which thymine (T), which is base 581 in the nucleotide sequence of SEQ ID NO: 6, is substituted with guanine (G), base 581 in the nucleotide sequence of SEQ ID NO: 6 In the nucleotide sequence in which inthymine (T) is substituted with cytosine (C), guanine (G), which is the 644th base in the nucleotide sequence of SEQ ID NO: 6, is replaced with adenine (A) Any one or more polynucleotides selected from the group consisting of a nucleotide sequence substituted with It may further include an agent that can be specifically detected.

As used herein, the term "agent capable of specifically detecting a protein" refers to 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 may be a GARS protein variant, for example, a specific compound or synthetic material targeting the p.P724H mutation site of the GARS protein variant, and more specifically specific to the GARS protein variant It may be a specific polypeptide, more specifically, it may be an antibody specific to a GARS protein variant, and more specifically, to a GARS protein variant consisting of an amino acid sequence in which proline, which is the 724th amino acid residue in the amino acid sequence of SEQ ID NO: 1, is substituted with histidine. It may be a specific antibody, but the type is not necessarily limited thereto. In addition, the agent capable of specifically detecting the protein may be a polypeptide specific for the twinkle protein mutant, GJB1 protein mutant, or SCN10A protein mutant, specifically an antibody specific for the protein mutant, but the type must be this It is not limited.

As used herein, the term “antibody” is a term known in the art and refers to a specific protein molecule directed against an antigenic site. The antibody specific for the protein variant is obtained by cloning the gene encoding the protein variant into an expression vector according to a conventional method to obtain a protein variant encoded by the gene, and can be prepared from the obtained protein variant by a conventional method. . This may also include a partial peptide that can be made from the protein variant, and the partial peptide may include at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The form of the antibody is not particularly limited, and as long as it has a polyclonal antibody, a monoclonal antibody, or antigen-binding property, a part thereof is also included in the antibody, and all immunoglobulin antibodies may be included. Furthermore, the antibody may also include a special antibody such as a humanized antibody.

The polyclonal antibody can be produced by a method well known in the art in which the above-described protein variant antigen is injected into an animal and blood is collected from the animal to obtain a serum containing the antibody. Such polyclonal antibodies can be prepared from hosts of any animal species, such as goats, rabbits, sheep, monkeys, horses, pigs, bovine dogs, and the like.

Monoclonal antibodies can be prepared using the hybridoma method well known in the art (Kohler and Milstein, European Journal of Immunology 6: 511-519, 1976), or a 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 using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.

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

As used herein, the term "agent capable of specifically detecting a polynucleotide" refers to 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 may be a substance that specifically binds to the GARS gene variant, specifically, a substance that specifically binds to the c.2171C>A mutation site of the GARS gene variant, , more specifically, may be a polynucleotide that specifically binds to the c.2171C>A mutation site of the GARS gene mutant, and more specifically, a primer or probe that specifically binds to the c.2171C>A mutation site of the GARS gene mutant. Or it may be an antisense nucleic acid, but the type is not necessarily limited thereto. In addition, the agent capable of specifically detecting the polynucleotide may be a substance that specifically binds to the TWNK gene mutant, GJB1 gene mutant, or SCN10A gene mutant, specifically for each mutation site of the gene mutant. It may be a polynucleotide that binds positively, more specifically, a primer, a probe, or an antisense nucleic acid, but the type is not necessarily limited thereto.

The agent capable of specifically detecting the polynucleotide includes a mutation site of the polynucleotide in each polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, 4, 6 or 8 or its complementary polynucleotide, and It may be a polynucleotide comprising 10 or more consecutive nucleotides selected from nucleotides.

An agent capable of specifically detecting the polynucleotide is one that exhibits a nucleotide mutation at a mutation site. Therefore, when one single-stranded polynucleotide is associated with the risk of developing Charcot-Marie-Tooth disease, the polynucleotide complementary to the single-stranded polynucleotide is naturally associated with the risk of developing Charcot-Marie-Tooth disease. can be judged. Accordingly, the composition according to an aspect may include a single-stranded polynucleotide associated with a risk of developing Charcot-Marie-Tooth disease having one specific sequence and a polynucleotide having a complementary sequence thereto. The agent capable of specifically detecting the polynucleotide can detect the c.2171C>A mutation in the polynucleotide of SEQ ID NO: 2. In addition, the agent capable of specifically detecting the polynucleotide includes c.1172G>A and c.1216C>T mutations in the polynucleotide of SEQ ID NO: 4, and c.53C>T, c. 112G>T, c.263C>T, c.371_372insAA, c.581T>G, c.581T>C, or c.644G>A mutation, or c.3749G>A mutation in the polynucleotide of SEQ ID NO: 8 can do.

The primer, probe or antisense nucleic acid specifically binds to a polynucleotide including a nucleotide sequence in which cytosine (C), which is the 2171th base in the nucleotide sequence of SEQ ID NO: 2, is substituted with adenine (A), and the base sequence of another nucleic acid material It may be one that does not specifically bind to. In addition, in the primer, probe or antisense nucleic acid, in the nucleotide sequence of SEQ ID NO: 4, guanine (G), the 1172th base, is substituted with adenine (A), and cytosine (C), the 1216th base, is substituted with thymine (T) A polynucleotide comprising a nucleotide sequence, in the nucleotide sequence of SEQ ID NO: 6, cytosine (C), the 53rd base, is substituted with thymine (T), or guanine (G), the 112th base, is substituted with thymine (T), 263 Cytosine (C), which is the second base, is substituted with thymine (T), two adenines (AA) are inserted between bases 371 and 372, or thymine (T), which is base 581, is substituted with guanine (G), or , a polynucleotide comprising a nucleotide sequence in which thymine (T) at the 581th base is substituted with cytosine (C), or guanine (G) at the 644th base is substituted with adenine (A), or the nucleotide sequence of SEQ ID NO: 8 It may be that it specifically binds to a polynucleotide including a nucleotide sequence in which guanine (G), the 3749th base, is substituted with adenine (A), and does not specifically bind to a nucleotide sequence of another nucleic acid material.

The primer, probe or antisense nucleic acid can be designed by a person skilled in the art based on each nucleotide sequence of the GARS gene variant, the TWNK gene variant, the GJB1 gene variant, or the SCN10A gene variant.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3' hydroxyl group, capable of forming a complementary template and base pair, and copying the template strand. It refers to 7 to 50 nucleic acid sequences serving as a starting point for Primers are usually synthesized but can also be used on 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 only if it is sufficiently complementary to hybridize with the template. Primers can initiate polymerization in an appropriate buffer and temperature, ie, DNA synthesis in the presence of reagents for DNA polymerase or reverse transcriptase and four different nucleoside triphosphates. PCR conditions, the length of the sense and antisense primers can be modified based on what is known in the art. Therefore, Charcot-Marie-Tooth disease can be diagnosed by PCR amplification using sense and antisense primers for each nucleotide sequence region of the GARS gene variant, TWNK gene variant, GJB1 gene variant, or SCN10A gene variant.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible to achieve specific binding to mRNA, and is labeled with a specific nucleic acid existence can be checked.

The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art. Accordingly, Charcot-Marie-Tooth disease can be diagnosed by performing hybridization using a probe complementary to each nucleotide sequence of the GARS gene variant, TWNK gene variant, GJB1 gene variant, or SCN10A gene variant.

The primer or probe may be chemically synthesized using a phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may incorporate additional features that do not change their basic properties. Examples of additional features that may be incorporated include, but are not limited to, methylation, encapsulation, substitution of one or more nucleic acids with homologs, and modifications between nucleic acids.

As used herein, the term "antisense nucleic acid" refers to a nucleic acid capable of forming a dimer with a GARS gene variant, TWNK gene variant, GJB1 gene variant, or SCN10A gene variant, which has a complementary sequence to the target. It means a molecule based on it, and can be used to detect each of the gene variants.

The antisense nucleic acid may be the polynucleotide or a fragment thereof, or complementary thereto. The fragments include 5 or more, specifically 10 or more, more specifically 10 to 1000, even more specifically 10 to 500, even more specifically 15 to 500, even more specifically 15 to 300 Dogs, more specifically, 15 to 200, more specifically, 15 to 150 nucleotides may be used, but an appropriate length may be selected in order to increase detection specificity.

Another aspect provides a kit for diagnosing whether or not the onset of Charcot-Marie-Tooth disease or the risk of the onset, comprising the composition.

The kit is a protein consisting of an amino acid sequence in which proline, which is the 724th amino acid residue in the amino acid sequence of SEQ ID NO: 1 from the test subject, is substituted with histidine, or cytosine (C), which is the 2171th base in the nucleotide sequence of SEQ ID NO: 2, is not an adenine (A By specifically detecting a polynucleotide comprising a nucleotide sequence substituted with ), it is possible to diagnose whether or not Charcot-Marie-Tooth disease occurs or the risk of developing it.

In addition, the kit is a protein consisting of an amino acid sequence in which arginine, the 391 amino acid residue in the amino acid sequence of SEQ ID NO: 3, is substituted with histidine, and arginine, the 406 amino acid residue, is not translated by a stop codon from the test subject, Or in the nucleotide sequence of SEQ ID NO: 4, a polynucleotide comprising a nucleotide sequence in which guanine (G), the 1172th base, is substituted with adenine (A), and cytosine (C), the 1216th base, is substituted with thymine (T) By detecting, it is possible to diagnose whether or not Charcot-Marie-Tooth disease develops or the risk of developing it.

In addition, the kit includes an amino acid sequence in which threonine, the 18th amino acid residue in the amino acid sequence of SEQ ID NO: 5, is substituted with isoleucine, and an amino acid in which valine, the 38th amino acid residue, in the amino acid sequence of SEQ ID NO: 5 is substituted with leucine, from the test subject Sequence, an amino acid sequence in which alanine, which is the 88th amino acid residue in the amino acid sequence of SEQ ID NO: 5, is substituted with valine, and lysine, which is the 124th amino acid residue in the amino acid sequence of SEQ ID NO: 5. An amino acid sequence in which the number of amino acids up to the last amino acid residue including, The amino acid sequence substituted with threonine, the amino acid sequence in which arginine, the 215th amino acid residue in the amino acid sequence of SEQ ID NO: 5, is substituted with glutamine, and the amino acid sequence in which the 1250th amino acid residue, arginine, is substituted with glutamine in the amino acid sequence of SEQ ID NO: 7 Any one or more proteins selected from the group consisting of each protein, or a nucleotide sequence in which cytosine (C), the 53rd base in the nucleotide sequence of SEQ ID NO: 6, is substituted with thymine (T), 112th in the nucleotide sequence of SEQ ID NO: 6 In the nucleotide sequence in which the base guanine (G) is substituted with thymine (T), the nucleotide sequence in which cytosine (C), the 263th base in the nucleotide sequence of SEQ ID NO: 6, is substituted with thymine (T), in the nucleotide sequence of SEQ ID NO: 6 A nucleotide sequence in which two adenines (AA) are inserted between bases 371 and 372, a nucleotide sequence in which thymine (T), the 581th base in the nucleotide sequence of SEQ ID NO: 6, is substituted with guanine (G), of SEQ ID NO: 6 A nucleotide sequence in which thymine (T), which is base 581 in the nucleotide sequence, is substituted with cytosine (C), guar, which is base 644 in the nucleotide sequence of SEQ ID NO: 6 A group consisting of each polynucleotide comprising a nucleotide sequence in which nin (G) is substituted with adenine (A), and a nucleotide sequence in which guanine (G), which is the 3749th base, is substituted with adenine (A) in the nucleotide sequence of SEQ ID NO: 8 By further detecting any one or more polynucleotides selected from Charcot-Marie-Tooth disease, it is possible to diagnose whether or not the onset of or the risk of the onset.

Specifically, the kit includes a p.P724H mutation site of a protein consisting of an amino acid sequence in which proline, which is the 724th amino acid residue in the amino acid sequence of SEQ ID NO: 1 in the test subject, is substituted with histidine, or base 2171 in the nucleotide sequence of SEQ ID NO: 2 Diagnosis of Charcot-Marie-Tooth disease or the risk of developing Charcot-Marie-Tooth disease by specifically detecting the c. can do. In addition, the kit can diagnose the onset or risk of Charcot-Marie-Tooth disease by further specifically detecting each mutation site of the TWNK gene mutant, GJB1 gene mutant, or SCN10A gene mutant in the test subject. .

The kit includes an antibody that specifically recognizes a protein consisting of an amino acid sequence in which proline, which is the 724th amino acid residue in the amino acid sequence of SEQ ID NO: 1, is substituted with histidine, or cytosine (C), which is the 2171th base in the nucleotide sequence of SEQ ID NO: 2 A primer, probe or antisense nucleic acid capable of specifically detecting a polynucleotide comprising the nucleotide sequence substituted with the adenine (A) may be included, and in addition thereto, one or more other component compositions, solutions or Devices may be included.

In addition, the kit includes a protein consisting of an amino acid sequence in which arginine, the 391 amino acid residue in the amino acid sequence of SEQ ID NO: 3, is substituted with histidine, and arginine, the 406 amino acid residue, is not translated by a stop codon and is terminated without translation, SEQ ID NO: 5 In the amino acid sequence, the 18th amino acid residue threonine is substituted with isoleucine, the 38th amino acid residue valine is substituted with leucine, the 88th amino acid residue alanine is substituted with valine, or the 124th amino acid residue lysine is the starting point A frameshift occurs so that the number of amino acids up to the last amino acid residue including lysine is 72, or methionine at the 194th amino acid residue is substituted with arginine, or methionine at the 194th amino acid residue is substituted with threonine, or 215 An antibody specifically recognizing a protein consisting of an amino acid sequence in which arginine, the first amino acid residue, is substituted with glutamine, or a protein consisting of an amino acid sequence in which arginine, the 1250 amino acid residue, in the amino acid sequence of SEQ ID NO: 7 is substituted with glutamine, or a sequence A polynucleotide comprising a nucleotide sequence in which guanine (G), the 1172th base, is substituted with adenine (A), and cytosine (C), the 1216th base, is substituted with thymine (T) in the nucleotide sequence of No. 4, SEQ ID NO: 6 In the nucleotide sequence of the nucleotide sequence, cytosine (C) at the 53rd base is substituted with thymine (T) ), two adenines (AA) are inserted between bases 371 and 372, thymine (T) at base 581 is substituted with guanine (G), or thymine (T) at base 581 is cytosine (C) or a polynucleotide comprising a nucleotide sequence in which guanine (G), which is the 644th base, is substituted with adenine (A), or guanine (G), which is the 3749th base in the nucleotide sequence of SEQ ID NO: 8, is not an adenine as (A) A primer, probe or antisense nucleic acid capable of specifically detecting a polynucleotide comprising the substituted nucleotide sequence may be further included.

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

The RT-PCR kit may include essential elements required for performing RT-PCR in order to specifically detect each mutation site of the gene variant. For example, the RT-PCR kit may contain, in addition to each pair of primers specific for each mutation site of the genetic variant, a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase. such as enzymes, DNase, RNase inhibitors, DEPC-water, sterile water, and the like.

The microarray chip may include a DNA or RNA polynucleotide probe. The microarray may include a configuration of a conventional microarray except for including a probe specific for each mutation site of the genetic variant. The microarray may provide useful information for diagnosing the possibility of developing Charcot-Marie-Tooth disease by detecting the presence or absence of each mutation site of the genetic variant.

A method for preparing a microarray by immobilizing a probe for each mutation site of the gene variant on a substrate is well known in the art. For example, the DNA microarray is, but not limited to, the c.539_544delGATTTG by a method using a micropipetting or a pin-type spotter using a piezoelectric method. A probe for the mutation site may be immobilized on a substrate. The substrate of the microarray may be coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde, but is not limited thereto. In addition, the substrate may be selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane, and nitrocellulose membrane, but is not limited thereto.

In addition, hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. In the detection, the nucleic acid sample is labeled with a fluorescent substance, for example, a label capable of generating a detectable signal including a substance such as Cy3 and Cy5, and then hybridized on a microarray, and a signal generated from the labeling substance By detecting the hybridization result can be detected.

The protein chip kit can measure the expression level of the protein variant. The protein chip kit may include a substrate, an appropriate buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent material, a chromogenic substrate, and the like for immunological detection of the antibody. As the substrate, a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from a polystyrene resin, a glass slide glass, etc. may be used, and peroxidase (peroxidase) may be used as the chromogenic enzyme. ), alkaline phosphatase, etc. may be used. In addition, FITC, RITC, etc. may be used as a fluorescent material, and ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (O-phenyl) as a color development substrate rendiamine), TMB (tetramethyl benzidine), and the like can be used.

In another aspect, in order to provide information for diagnosing Charcot-Marie-Tooth disease or predicting the risk of onset, in a biological sample isolated from an individual, proline, the 724th amino acid residue in the amino acid sequence of SEQ ID NO: 1, is substituted with histidine Provided is a method for detecting a protein consisting of an amino acid sequence or a polynucleotide including a nucleotide sequence in which cytosine (C), which is the 2171th base in the nucleotide sequence of SEQ ID NO: 2, is substituted with adenine (A).

In the method, in a biological sample isolated from a subject, in the amino acid sequence of SEQ ID NO: 3, arginine, the 391th amino acid residue, is substituted with histidine, and the 406th amino acid residue, arginine, consists of an amino acid sequence that is not translated by a stop codon and is terminated A polynucleotide comprising a protein or a nucleotide sequence in which guanine (G), the 1172th base, is substituted with adenine (A), and cytosine (C), the 1216th base, is substituted with thymine (T) in the nucleotide sequence of SEQ ID NO: 4 may be further detected.

In the method, in a biological sample isolated from an individual, in the amino acid sequence of SEQ ID NO: 5, threonine, the 18th amino acid residue, is substituted with isoleucine, and in the amino acid sequence of SEQ ID NO: 5, valine, the 38th amino acid residue, is substituted with leucine The amino acid sequence, the amino acid sequence in which alanine, the 88th amino acid residue in the amino acid sequence of SEQ ID NO: 5, is substituted with valine, and lysine, the 124th amino acid residue in the amino acid sequence of SEQ ID NO: 5, as the starting point, a frameshift occurs. An amino acid sequence in which the number of amino acids up to the last amino acid residue, including lysine, is 72; The amino acid sequence in which methionine is substituted with threonine, the amino acid sequence in which arginine, the 215th amino acid residue in the amino acid sequence of SEQ ID NO: 5, is substituted with glutamine, and the amino acid in which the 1250th amino acid residue, arginine, is substituted in the amino acid sequence of SEQ ID NO: 7 Any one or more proteins selected from the group consisting of each protein consisting of a sequence, or a nucleotide sequence in which cytosine (C), which is the 53rd base in the nucleotide sequence of SEQ ID NO: 6, is substituted with thymine (T), in the nucleotide sequence of SEQ ID NO: 6 A nucleotide sequence in which guanine (G) at the 112th base is substituted with thymine (T), a nucleotide sequence in which cytosine (C) at base 263 is substituted with thymine (T) in the nucleotide sequence of SEQ ID NO: 6, the nucleotide sequence of SEQ ID NO: 6 A nucleotide sequence in which two adenines (AA) are inserted between bases 371 and 372 in the sequence, a nucleotide sequence in which thymine (T), which is base 581 in the nucleotide sequence of SEQ ID NO: 6, is substituted with guanine (G), SEQ ID NO: A nucleotide sequence in which thymine (T), which is the 581th base in the nucleotide sequence of 6, is substituted with cytosine (C), 644th in the nucleotide sequence of SEQ ID NO: 6 Each polynucleotide comprising a nucleotide sequence in which guanine (G), a base, is substituted with adenine (A), and a nucleotide sequence in which guanine (G), which is the 3749th base, is substituted with adenine (A) in the nucleotide sequence of SEQ ID NO: 8 It may be to further detect any one or more polynucleotides selected from the group consisting of.

As used herein, the term “individual” refers to an individual who wants to determine whether or not the onset or possibility of Charcot-Marie-Tooth disease or the risk of developing the disease is predicted. The subject may be a vertebrate, specifically mammals, amphibians, reptiles, birds, etc., may be more specifically a mammal, for example, a human ( Homo sapiens ).

As used herein, the term "biological sample" may include, but is not limited to, a sample such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine isolated from an individual. Methods for obtaining a biological sample from a subject are known in the art. In the case of a protein sample, isolated tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine may be directly used, or a protein dissolved in a lysis buffer may be used. The protein sample includes not only the purely isolated protein but also the crudely isolated protein, for example, a cell lysate containing the protein. In the case of a nucleic acid sample, the nucleic acid can be directly isolated from tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, or a nucleic acid in which a specific region is amplified by a nucleic acid amplification method such as PCR can be used. 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 purely isolated nucleic acids, but also crudely isolated nucleic acids, for example, cell lysates containing nucleic acids.

The method may be to directly determine and detect the amino acid residue of the amino acid mutation site in the biological sample. Methods for determining amino acid residues are known in the art. For example, a protein sequencer can be used to directly determine the amino acid residues at the site of amino acid variation.

The method may be to detect the protein using an agent capable of specifically detecting the protein. Specifically, the agent capable of specifically detecting the protein may be an antibody, but is not limited thereto.

Methods for detecting the protein include Western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS and an assay selected from the group consisting of a protein chip, but is not limited thereto.

The method may be to directly determine and detect the nucleotide of the polynucleotide mutation site in the biological sample. Methods for determining nucleotides are known in the art. For example, a nucleotide sequencer may be used to directly determine the nucleotide of a polynucleotide variation site. The nucleotide of a mutation site can be directly determined by a nucleotide sequencing method of a known nucleic acid. Methods for determining nucleotide sequencing may include Sanger (or dideoxy) sequencing methods, next-generation sequencing, next-generation sequencing, or Maxam-Gilbert (chemical cleavage) methods.

The method may be to detect the polynucleotide using an agent capable of specifically detecting the polynucleotide. Specifically, the agent capable of specifically detecting the polynucleotide may be a primer, a probe, or an antisense nucleic acid, but is not limited thereto.

The method for detecting the polynucleotide may be to use an assay selected from the group consisting of reverse transcription polymerase reaction, competitive reverse transcription polymerase reaction, real-time reverse transcription polymerase reaction, RNase protection assay (RPA), Northern blotting and DNA chip. However, the present invention is not limited thereto.

In the method, p.P724H mutation of a protein consisting of an amino acid sequence in which proline, which is the 724th amino acid residue in the amino acid sequence of SEQ ID NO: 1, is substituted with histidine, or at position 2171 in the nucleotide sequence of SEQ ID NO: 2 in a biological sample isolated from an individual When the c.2171C>A mutation of a polynucleotide including a nucleotide sequence in which cytosine (C) is replaced with adenine (A) is present, it can be diagnosed as having or at risk of Charcot-Marie-Tooth disease. .

In addition, in the method, in the biological sample isolated from the subject, in the amino acid sequence of SEQ ID NO: 3, arginine, the 391th amino acid residue, is substituted with histidine, and the 406th amino acid residue, arginine, is untranslated by a stop codon. In the p.R391H and p.R406X mutations of a protein consisting of ), when c.1172G>A and c.1216C>T mutations of a polynucleotide comprising a nucleotide sequence substituted with

In addition, in the method, in the biological sample isolated from the subject, in the amino acid sequence of SEQ ID NO: 5, threonine, which is the 18th amino acid residue, is substituted with isoleucine, or the 38th amino acid residue, valine, is substituted with leucine, or the 88th amino acid residue is Alanine is substituted with valine, or a frameshift occurs starting from lysine, the 124th amino acid residue, so that the number of amino acids up to the last amino acid residue including the lysine becomes 72, or methionine, the 194th amino acid residue, is converted to arginine p.T18I, p.V38L, p.A88V, p.K124fs*72 of a protein consisting of an amino acid sequence in which the amino acid sequence is substituted, methionine at the 194th amino acid residue is substituted with threonine, or arginine at the 215th amino acid residue is substituted with glutamine , p.M194R, p.M194T, or p.R215Q mutation, or cytosine (C) at the 53rd base in the nucleotide sequence of SEQ ID NO: 6 is substituted with thymine (T), or guanine (G) at the 112th base is thymine (T), cytosine (C) at base 263 is substituted with thymine (T), two adenines (AA) are inserted between bases 371 and 372, or thymine (T) at base 581 A polynucleotide comprising a nucleotide sequence in which this guanine (G) is substituted, thymine (T) at the 581th base is substituted with cytosine (C), or guanine (G) at the 644th base is substituted with adenine (A) Charcot-Marie when more c.53C>T, c.112G>T, c.263C>T, c.371_372insAA, c.581T>G, c.581T>C, or c.644G>A mutations of -Can be diagnosed as having or at risk of developing Tooth disease.

In addition, in the method, the SCN10A protein variant consisting of an amino acid sequence in which arginine, which is the 1250 amino acid residue in the amino acid sequence of SEQ ID NO: 7 in the amino acid sequence of SEQ ID NO: 7, is substituted with glutamine, or base 3749 in the nucleotide sequence of SEQ ID NO: 8 in the biological sample isolated from the subject The p.R1250Q mutation of a protein consisting of a nucleotide sequence in which phosphorus guanine (G) is substituted with adenine (A), or a nucleotide sequence in which guanine (G), which is the 3749th base in the nucleotide sequence of SEQ ID NO: 8, is substituted with adenine (A) When the c.3749G>A mutation of the polynucleotide comprising

"Risk of developing Charcot-Marie-Tooth disease" may be a relative risk of developing Charcot-Marie-Tooth disease. For example, the risk of developing the disease may indicate whether the probability of the disease is increased as compared to a healthy normal group or a group having a reference genome sequence. In addition, the risk of occurrence may indicate whether an individual having a specific allele or genotype has an increased probability of developing Charcot-Marie-Tooth disease compared to an individual having another specific allele or genotype.

The GARS protein variant or GARS gene variant according to an aspect may be used as a marker for diagnosing Charcot-Marie-Tooth disease. Diagnosis using the GARS gene mutant enables accurate early diagnosis of Charcot-Marie-Tooth disease through genetic testing, and accordingly, treatment effects that have been recently developed can be applied early to maximize the therapeutic effect.

1 is a pedigree of a family including a patient with Charcot-Marie-Tooth disease with a GARS gene mutation c.2171C>A. The white figure means normal, and the black figure means a patient with Charcot-Marie-Tooth disease. An asterisk (*) indicates an individual for which a gene mutation was investigated, and an arrow (↗) indicates a proband.
2 is a nucleotide sequence chromatogram including a GARS gene mutation site of a family including a patient with Charcot-Marie-Tooth disease having a GARS gene mutation c.2171C>A.
Figure 3 is a vertebrate species of human, rhesus monkey (Rhesus monkey), rat, dog, elephant, chicken, African frog ( Xenopus tropicalis ), zebrafish and lamprey (Lamprey) of the mutation site and surrounding amino acid sequence of the GARS protein. Conservation analysis results.
4 is a pedigree of a family including patients with Charcot-Marie-Tooth disease with TWNK gene mutations c.1172G>A and c.1216C>T. The white figure means normal, and the black figure means a patient with Charcot-Marie-Tooth disease. An asterisk (*) indicates an individual for which a gene mutation was investigated, and an arrow (↗) indicates a proband. The genotype of the variant is indicated below the individual.
5 is a nucleotide sequence chromatogram including the TWNK gene mutation site of a family including a patient with Charcot-Marie-Tooth disease having TWNK gene mutations c.1172G>A and c.1216C>T.
Figure 6 is a mutation site of the twinkle protein and surrounding amino acid sequences in vertebrate species human, Rhesus monkey, rat, dog, elephant, chicken, African frog ( Xenopus tropicalis ), zebrafish and lamprey (Lamprey). Conservation analysis results.
7 is a view showing the analysis results of mitochondrial DNA deletion (mtDNA deletion) of a family including a patient with Charcot-Marie-Tooth disease having TWNK gene mutations c.1172G>A and c.1216C>T (Ctrl(WT)) : Normal control; Ctrl (Del): positive control lacking mitochondrial DNA; FC868-1: patient himself; FC868-2: patient's father; and FC868-3: patient's mother).
8 is a view showing the analysis results of mitochondrial DNA depletion (mtDNA depletion) of a family including a patient with Charcot-Marie-Tooth disease having TWNK gene mutations c.1172G>A and c.1216C>T (A: the patient himself) (FC868-1); B: Patient's father (FC868-2); and C: Patient's mother (FC868-3)).
9 shows GJB1 gene mutations c.53C>T (FIG. 9A), c.112G>T (FIG. 9B), c.263C>T (FIG. 9C), c.371_372insAA (FIG. 9D), c.581T>G ( Fig. 9E), or a pedigree of families including patients with Charcot-Marie-Tooth disease with c.581T>C (Fig. 9F). The white figure means normal, and the black figure means a patient with Charcot-Marie-Tooth disease. An asterisk (*) indicates an individual for which a gene mutation was investigated, and an arrow (↗) indicates a proband.
10 is a nucleotide sequence chromatogram including a GJB1 gene mutation site of a family including a patient with Charcot-Marie-Tooth disease having a GJB1 gene mutation c.53C>T.
11 is a nucleotide sequence chromatogram including a GJB1 gene mutation site of a family including a patient with Charcot-Marie-Tooth disease having a GJB1 gene mutation c.112G>T.
12 is a nucleotide sequence chromatogram including a GJB1 gene mutation site of a family including a patient with Charcot-Marie-Tooth disease having a GJB1 gene mutation c.263C>T.
13 is a nucleotide sequence chromatogram including a GJB1 gene mutation site of a family including a patient with Charcot-Marie-Tooth disease having the GJB1 gene mutation c.371_372insAA.
14 is a nucleotide sequence chromatogram including a GJB1 gene mutation site of a family including a patient with Charcot-Marie-Tooth disease having a GJB1 gene mutation c.581T>G.
15 is a nucleotide sequence chromatogram including a GJB1 gene mutation site of a family (myself) including a patient with Charcot-Marie-Tooth disease having a GJB1 gene mutation c.581T>C.
16 is a nucleotide sequence chromatogram including a GJB1 gene mutation site of a family (myself) including a patient with Charcot-Marie-Tooth disease having a GJB1 gene mutation c.644G>A.
17 is a mutation of the GJB1 protein and surrounding amino acid sequences in vertebrate species human, Rhesus monkey, rat, dog, elephant, chicken, African frog ( Xenopus tropicalis ), zebrafish and lamprey (Lamprey). Conservation analysis results (Fig. 17A: p.T18I mutation site; Fig. 17B: p.V38L mutation site; Fig. 17C: p.A88V mutation site; Fig. 17D: p.K124fs*72 mutation site; Fig. 17E: p.M194R and p.M194T mutation site; and Figure 17F: p.R215Q mutation site;).
18 is a pedigree diagram of a family including a patient with Charcot-Marie-Tooth disease carrying the SCN10A gene mutation c.3749G>A. The white figure means normal, and the black figure means a patient with Charcot-Marie-Tooth disease. An asterisk (*) indicates an individual for which a gene mutation was investigated, and an arrow (↗) indicates a proband.
19 is a nucleotide sequence chromatogram including the SCN10A gene mutation site of a family (myself) including a patient with Charcot-Marie-Tooth disease having the SCN10A gene mutation c.3749G>A.
20 is a result of conservation analysis of the mutation site and surrounding amino acid sequences of the SCN10A protein in vertebrate species human ( Homo sapiens ), rat ( Mus musculus ), cattle ( Bos taurus ), and wolf ( Canis lupus ).

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1. Charcot-Marie-Tooth disease-associated GARS gene and protein mutation confirmation

Example 1-1. Selection of test subjects

Genetic mutations were investigated in families including patients with Charcot-Marie-Tooth disease (Family ID: FC855), and the control group was 500 normal subjects.

Example 1-2. Identification of causative genes for Charcot-Marie-Tooth disease

The causative gene was identified in a patient with Charcot-Marie-Tooth disease among blood samples of the test subject family selected in Example 1-1.

Specifically, peripheral blood was obtained from the family member, and genomic DNA was extracted using the QIAamp blood DNA purification kit (Qiagen, Hilden, Germany), followed by an automatic sequencing analyzer ABI3130XL and Genemapper software (Life Technologies, Foster City, CA, USA) was used for genotyping. In addition, whole exomes were captured using the SureSelect Human All Exon 50M kit (Agilent Technologies, Santa Clara, CA, USA), followed by standard pair-ends using a HiSeq2000 Genome Analyzer (Illumina, San Diego, CA, USA). Sequencing was performed according to the sequencing protocol in (paired-end) mode. Whole exome sequencing (WES) was performed at Macrogen (Seoul, Korea). UCSC assembly hg19 was used as a reference sequence.

Small nucleotide variants (SNVs) were extracted using the GATK program, and the variants were annotated using the ANNOVAR program. Sequences were extracted only when the read depth was 5 or more. Annotated SNVs were compared to variants registered in dbSNP150 (http://www.ncbi.nlm.nih.gov).

Potential functionally significant mutations (missense, nonsense, exonic indel, stop gain and splicing sites) were first identified from the data. was selected. Then, new or rare variants (minor allele frequency (MAF) less than 0.01) were further filtered using the 1000 Genomes Project (http://www.1000genomes.org/). Candidate causative variants were identified by the Sanger sequencing method using the ABI3130XL genetic analyzer. MUpro (http://www.ics.uci.edu/~baldig/mutation), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2), and SIFT (http://sift.jcvi. org), an in silico analysis was performed. Genomic evolutionary rate profiling (GERP) scores were determined using the GERP++ program (http://mendel.stanford.edu/SidowLab/downloads/gerp/index.html).

As a result of the analysis, as shown in Table 1 and FIG. 1 , it was confirmed that the c.2171C>A mutation of the GARS gene was present in an individual exhibiting the Charcot-Marie-Tooth disease phenotype among the test subject families.

number gender/age GARS gene c.2171C>A mutation phenotype FC855-1 (myself) M/12 C/ A CMT2D FC855-2 (Mo) female/45 C/C - FC855-3 (minor) Male/43 C/C -

In addition, as shown in Table 2, the c.2171C>A (p.P724H) mutation of the GARS gene was confirmed as the causative gene mutation of Charcot-Marie-Tooth disease from the whole exome sequencing result, which was dbSNP150, 1000 genomes. It was confirmed that it was a new mutation unknown to the project or the like.

transition Nucleotides (Nt) c.2171C>A Amino acids (AA) p.P724H heredity Autosomal dominant (AD) human genome database 1000G (Oct 2011) - In silico analysis dbSNP150 - GERP 4.72 SIFT 0.02 PolyPhen2 0.844 Mupro -0.923

For the GARS gene of the family selected in Example 1-1, including a patient with Charcot-Marie-Tooth disease, the base at the mutation site was confirmed by the Sanger sequencing method.

As a result, as shown in FIG. 2 , it was confirmed that the c.2171C>A mutation was present in the patient's own GARS gene, unlike normal parents.

Examples 1-3. Conservative region analysis at mutation sites

By aligning the amino acid sequence of the mutation site and the surrounding region, it was confirmed whether the sequence was conserved between vertebrates. Vertebrate species were humans, Rhesus monkeys, rats, dogs, elephants, chickens, African frogs ( Xenopus tropicalis ), zebrafish and lampreys.

As a result, as shown in FIG. 3 , it was confirmed that the mutation site of the GARS protein was located in a region conserved at a high level among vertebrate species.

Example 2. Charcot-Marie-Tooth disease-associated TWNK gene and protein mutation confirmation

Example 2-1. Selection of test subjects

Genetic mutations were investigated in families including patients with Charcot-Marie-Tooth disease (Family ID: FC868), and the control group was 500 normal subjects.

Example 2-2. Identification of causative genes for Charcot-Marie-Tooth disease

The causative gene of Charcot-Marie-Tooth disease was identified in the same manner as in Example 1-2 using the blood sample of the test subject family selected in Example 2-1.

As a result of the analysis, as shown in Table 3 and FIG. 4, it was confirmed that both c.1172G>A and c.1216C>T mutations of the TWNK gene were present in individuals exhibiting the Charcot-Marie-Tooth disease phenotype among the test subject families. did

number gender/age Whether the TWNK gene is mutated phenotype c.1172G>A c.1216C>T FC868-1 (myself) female/10 G/ A C/ T CMT2 FC868-2 (minor) Male/43 G/G C/ T - FC868-3 (Mo) female/41 G/ A C/C - FC868-4 (younger brother) M/12 G/G C/C -

In addition, as shown in Table 4, from the result of whole exome sequencing, Charcot-Marie-Tooth disease causative gene mutation, c.1172G >A (p.R391H) mutation and c.1216C>T (p. R406X) mutation was confirmed, and among them, the c.1216C>T mutation was confirmed to be a novel mutation unknown to dbSNP150 and 1000 Genome Project.

transition Nucleotides (Nt) c.1172G>A c.1216C>T Amino acids (AA) p.R391H p.R406X heredity Autosomal recessive (AR) human genome database 1000G (Oct 2011) 0.0002 - In silico analysis dbSNP150 rs556445621 - GERP 3.19 6.04 SIFT 0.05 - PolyPhen2 1.000 - Mupro -0.49 -

For the TWNK gene of the family selected in Example 2-1, including a patient with Charcot-Marie-Tooth disease, the base at the mutation site was confirmed by the Sanger sequencing method.

As a result, as shown in FIG. 5 , unlike a parent who has only one of the TWNK gene mutations c.1172G>A and c.1216C>T mutations or a younger brother who does not have all of the above mutations, the patient himself/herself has the above two mutations. It was confirmed that they had all of them.

Example 2-3. Conservative region analysis at mutation sites

By aligning the amino acid sequence of the mutation site and the surrounding region, it was confirmed whether the sequence was conserved between vertebrates. Vertebrate species were humans, Rhesus monkeys, rats, dogs, elephants, chickens, African frogs ( Xenopus tropicalis ), zebrafish and lampreys.

As a result, as shown in FIG. 6 , it was confirmed that the mutation site of the twinkle protein was located in a region conserved at a high level among vertebrate species.

Example 2-4. Confirmation of Mitochondrial DNA Defects

Mitochondrial DNA 4977bp common deletion (8469-13447) targeting the gene of the patient himself (FC868-1), father (FC868-2) and mother (FC868-3) among the family members selected in Example 2-1 Analysis was performed, a normal sample was used as a control, and a sample lacking mitochondrial DNA was used as a positive control.

Specifically, Long-PCR was performed using a PCR system (Expand 20kb plus PCR system; Roche) with forward primers (8182-8208) and reverse primers (13981-13957). PCR system using 2 μl of 10-20 ng of mitochondrial DNA, 2 μl of 10 pmoles of each primer, 1 μl of 10 mM dNTPs mix, 2 μl of 10x reaction buffer, and 0.4 μl of 5 units of enzyme mix so that the total reaction solution becomes 20 μl (GeneAmp PCR system 9700; Life Technology) was amplified. 1.2% agarose gel electrophoresis was performed on the amplified product to confirm the result.

As a result, as shown in FIG. 7 , no mitochondrial DNA defects were observed in both Charcot-Marie-Tooth disease patients and normal family members with both c.1172G>A and c.1216C>T mutations of the TWNK gene.

Example 2-5. Check whether mitochondrial DNA is reduced

Among the families selected in Example 2-1, real-time quantitative PCR (real-time qPCR) was performed to confirm the mitochondrial DNA reduction of the patient himself (FC868-1), father (FC868-2) and mother (FC868-3). , a normal sample was used as a control.

Specifically, the PCR reaction was performed with a PCR system (CFX96® Real-Time PCR Detection System; Bio-Rad Laboratories, USA) using SYBR Green Real-Time PCR Mix (Toyobo Co.). To confirm the expression of mitochondrial DNA, ND1 (NADH-ubiquinone oxidoreductase chain 1) and COX1 (Cytochrome c oxidase I), a pair of forward and reverse primers for each gene were used. It was created and used based on The PCR reaction was performed by repeating 40 cycles of initial denaturation at 95° C. for 1 minute, followed by a reaction at 95° C. for 15 seconds, 56° C. for 15 seconds, and 72° C. for 45 seconds.

As a result, as shown in FIG. 8, the expression levels of both mitochondrial DNAs were significantly reduced in Charcot-Marie-Tooth disease patients having both c.1172G>A and c.1216C>T mutations of the TWNK gene, and the mutations In parents (FC868-2 and FC868-3) having only one mutation, a weak level of expression was confirmed. Through this, it was confirmed that the mitochondrial DNA expression level was decreased due to the mutation of the TWNK gene, which is thought to be involved in mitochondrial DNA replication.

Example 3. Charcot-Marie-Tooth disease-associated GJB1 gene and protein mutation confirmation

Example 3-1. Selection of test subjects

Genetic mutations were investigated in families including patients with Charcot-Marie-Tooth disease (Family ID: FC1051, FC905, FC934, FC997, FC922, and FC977), and the control group was 500 normal people.

Example 3-2. Identification of causative genes for Charcot-Marie-Tooth disease

The causative gene of Charcot-Marie-Tooth disease was identified in the same manner as in Example 1-2 using the blood sample of the test subject family selected in Example 3-1, and PROVEAN (http:/ /provean.jcvi.org) of the prediction algorithm was further used.

As a result of the analysis, as shown in Tables 5 and 9, c.53C>T mutation, c.112G>T mutation, c.263C>T of the GJB1 gene in individuals exhibiting the Charcot-Marie-Tooth disease phenotype among the test subject families. It was confirmed that the mutation, c.371_372insAA mutation, c.581T>G mutation, c.581T>C mutation, or c.644G>A mutation existed.

GJB1 gene mutation number gender/age mutation or not phenotype c.53C>T FC1051-1 (myself) Male/24 T CMTX1 FC1051-2 (minor) Male/48 C - c.112G>T FC905-1 (myself) Male/45 T CMTX1 FC905-2 (daughter) Female/17 T /G CMTX1 FC905-3 (son) M/14 G - FC905-4 (Mo) T /G CMTX1 c.263C>T FC934-1 (myself) female/59 C/ T CMTX1 FC934-2 (younger brother) female/56 C/ T CMTX1 FC934-3 (daughter) female/33 C/C - FC934-4 (son) M/30 T CMTX1 c.371_372insAA FC997-1 (myself) female/26 -/ AA CMTX1 FC997-2 (minor) M/55 --- - FC997-3 (Mother) female/52 --- - c.581T>G FC922-1 (myself) M/19 G CMTX1 FC922-2 (younger brother) M/15 T - FC922-3 (parent) female/44 T/T - c.581T>C FC977-1 (myself) M/55 C CMTX1 c.644G>A FC839-1 (myself) female/46 G/ A CMTX1

In addition, as shown in Table 6, from the whole exome sequencing result, Charcot-Marie-Tooth disease causative gene mutation, c.53C >T (p.T18I) mutation of GJB1 gene, c.112G>T (p. V38L) mutation, c.263C>T (p.A88V) mutation, c.371_372insAA (p.K124fs*72) mutation, c.581T>G (p.M194R) mutation, c.581T>C (p.M194T) A mutation, or c.644G>A (p.R215Q) mutation, was identified, all of which were novel mutations unknown to the 1000 Genome Project, c.53C>T mutation, c.112G>T mutation, c.263C >T mutation, c.371_372insAA mutation, c.581T>G mutation, and c.581T>C mutation were confirmed to be novel mutations unknown to dbSNP150.

GJB1 gene mutation human genome database In silico analysis Nucleotides (Nt) amino acid
(AA) 1000G (Oct 2011) dbSNP150 GERP PROVEAN SIFT PolyPhen2 Mupro c.53C>T p.T18I - - 4.36 -5.789 - One 0.70 c.112G>T p.V38L - - 4.26 - 0 0.443 0.32 c.263C>T p.A88V - - 4.26 - - One 0.30 c.371_372insAA p.K124fs*72 - - 4.67 - - - - c.581T>G p.M194R - - 4.99 - 0 One -0.515 c.581T>C p.M194T - - 4.99 -5.637 One -One c.644G>A p.R215Q - rs864622215 4.9 0.03 0.075 -One

With respect to the GJB1 gene of the family selected in Example 3-1, including patients with Charcot-Marie-Tooth disease, the bases at each mutation site were confirmed by the Sanger sequencing method.

As a result, as shown in FIGS. 10 to 16 , it was confirmed that there was no mutation in the normal part of the FC1051 family, but only in the patient himself, and the normal son in the FC905 family had no mutation, but the patient himself, mother, and daughter. In the FC934 family, it was confirmed that the mutation was present in the patient, brother, and son, and the normal daughter did not have the mutation. In the FC922 family, the normal mother and brother did not have mutations, but the mutation was confirmed only in the patient himself.

Example 3-3. Conservative region analysis at mutation sites

For each mutation of the GJB1 gene, the amino acid sequence of the mutation site and the surrounding region was arranged to confirm whether the sequence was conserved among vertebrates. Vertebrate species were humans, Rhesus monkeys, rats, dogs, elephants, chickens, African frogs ( Xenopus tropicalis ), zebrafish and lampreys.

As a result, as shown in FIG. 17 , it was confirmed that the mutation site of the GJB1 protein was located in a region conserved at a high level among vertebrate species.

Example 4. Charcot-Marie-Tooth disease-associated SCN10A gene and protein mutation confirmation

Example 4-1. Selection of test subjects

Genetic mutations were investigated in families including patients with Charcot-Marie-Tooth disease (Family ID: FC1078), and the control group was 500 normal subjects.

Example 4-2. Identification of causative genes for Charcot-Marie-Tooth disease

The causative gene of Charcot-Marie-Tooth disease was identified in the same manner as described in Example 1-2 using the blood sample of the test subject family selected in Example 4-1, and in silico analysis, PROVEAN (http The prediction algorithm of ://provean.jcvi.org) was used.

.

As a result of the analysis, as shown in Table 7 and FIG. 18 , it was confirmed that the c.3749G>A mutation of the SCN10A gene was present in the individuals exhibiting the Charcot-Marie-Tooth disease phenotype among the test subject families.

number gender/age SCN10A gene c.3749G>A mutation phenotype FC1078-1 (myself) Male/40 A CMT1

In addition, as shown in Table 8, the c.3749G>A (p.R1250Q) mutation of the SCN10A gene was confirmed as the causative gene mutation of Charcot-Marie-Tooth disease from the result of whole exome sequencing, which was applied to the 1000 Genome Project. It was confirmed that it was an unknown novel mutation.

transition Nucleotides (Nt) c.3749G>A Amino acids (AA) p.R1250Q heredity Autosomal dominant (AD) human genome database 1000G (Oct 2011) - In silico analysis dbSNP150 rs774893568 GERP 4.23 PROVEAN -3.481 PolyPhen2 0.987 Mupro 0.041

With respect to the SCN10A gene of the family selected in Example 4-1, including patients with Charcot-Marie-Tooth disease, the base at the mutation site was confirmed by the Sanger sequencing method.

As a result, as shown in FIG. 19 , it was confirmed that the c.3749G>A mutation was present in the patient's own SCN10A gene.

Example 4-3. Conservative region analysis at mutation sites

By aligning the amino acid sequence of the mutation site and the surrounding region, it was confirmed whether the sequence was conserved between vertebrates. Vertebrate species were humans ( Homo sapiens ), rats ( Mus musculus ), cattle ( Bos taurus ), and wolves ( Canis lupus ).

As a result, as shown in FIG. 20 , it was confirmed that the mutation site of the SCN10A protein was located in a region conserved at a high level among vertebrate species.

<110> Samsung Life Public Welfare Foundation KONGJU NATIONAL UNIVERSITY INDUSTRY-UNIVERSITY COOPERATION FOUNDATION Dong-A University Research Foundation For Industry-Academy Cooperation <120> Marker for diagnosing Charcot-Marie-Tooth disease and use thereof <130> PN130969KR <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 739 <212> PRT <213> Homo sapiens <400> 1 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 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 2 60 265 270 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 T yr 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 A rg 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 G lu 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 < 210> 2 <211> 2220 <212> DNA <213> Homo sapiens <400> 2 atgccctctc cgcgtccagt gctgcttaga ggtgctcgcg ccgctctgct gctgctgctg 60 ccgccccggc tcttagcccg accctcgctc ctgctccgcc ggtccctcag cgcggcctcc 120 tgccccccga tctccttgcc cgccgccgcc tcccggagca gcatggacgg cgcgggggct 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 accatgct ca 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 tcggaaaatg 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 agaacccaaa 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 2220 <210> 3 <211> 684 <212 > PRT <213> Homo sapiens <400> 3 Met Trp Val Leu Leu Arg Ser Gly Tyr Pro Leu Arg Ile Leu Leu Pro 1 5 10 15 Leu Arg Gly Glu Trp Met Gly Arg Arg Gly Leu Pro Arg Asn Leu Ala 20 25 30 Pro Gly Pro Pro Arg Arg Arg Tyr Arg Lys Glu Thr Leu Gln Ala Leu 35 40 45 Asp Met Pro Val Leu Pro Val Thr Ala Thr Glu Ile Arg Gln Tyr Leu 50 55 60 Arg Gly His Gly Ile Pro Phe Gln Asp Gly His Ser Cys Leu Arg Ala 65 70 75 80 Leu Ser Pro Phe Ala Glu Ser Ser Gln Leu Lys Gly Gln Thr Gly Val 85 90 95 Thr Thr Ser Phe Ser Leu Phe Ile Asp Lys Thr Thr Gly His Phe Leu 100 105 110 Cys Met Thr Ser Leu Ala Glu Gly Ser Trp Glu Asp Phe Gln Ala Ser 115 120 125 Val Glu Gly Arg Gly Asp Gly Ala Arg Glu Gly Phe Leu Leu Ser Lys 130 135 140 Ala Pro Glu Phe Glu Asp Ser Glu Glu Val Arg Arg Ile Trp Asn Arg 145 150 155 160 Ala Ile Pro Leu Trp Glu Leu Pro Asp Gln Glu Glu Val Gln Leu Ala 165 170 175 Asp Thr Met Phe Gly Leu Thr Lys Val Thr Asp Asp Thr Leu Lys Arg 180 185 190 Phe Ser Val Arg Tyr Leu Arg Pro Ala Arg Ser Leu Val Phe Pro Trp 195 200 205 Phe Ser Pro Gly Gly Ser Gly Leu Arg Gly Leu Lys Leu Leu Glu Ala 210 215 220 Lys Cys Gln Gly Asp Gly Val Ser Tyr Glu Glu Thr Thr Ile Pro Arg 225 230 235 240 Pro Ser Ala Tyr His Asn Leu Phe Gly Leu Pro Leu Ile Ser Arg Arg 245 250 255 Asp Ala Glu Val Val Leu Thr Ser Arg Glu Leu Asp Ser Leu Ala Leu 260 265 270 Asn Gln Ser Thr Gly Leu Pro Thr Leu Thr Leu Pro Arg Gly Thr Thr 275 280 285 Cys Leu Pro Pro Ala Leu Leu Pro Tyr Leu Glu Gln Phe Arg Arg Ile 290 295 300 Val Phe Trp Leu Gly Asp Asp Leu Arg Ser Trp Glu Ala Ala Lys Leu 305 310 315 320 Phe Ala Arg Lys Leu Asn Pro Lys Arg Cys Phe Leu Val Arg Pro Gly 325 330 335 Asp Gln Gln Pro Arg Pro Leu Glu Ala Leu Asn Gly Gly Phe Asn Leu 340 345 350 Ser Arg Ile Leu Arg Thr Ala Leu Pro Ala Trp His Lys Ser Ile Val 355 360 365 Ser Phe Arg Gln Leu Arg Glu Glu Val Leu Gly Glu Leu Ser Asn Val 370 375 380 Glu Gln Ala Ala Gly Leu Arg Trp Ser Arg Phe Pro Asp Leu Asn Arg 385 390 395 400 Ile Leu Lys Gly His Arg Lys Gly Glu Leu Thr Val Phe Thr Gly Pro 405 410 415 Thr Gly Ser Gly Lys Thr Thr Phe Ile Ser Glu Tyr Ala Leu Asp Leu 420 425 430 Cys Ser Gln Gly Val Asn Thr Leu Trp Gly Ser Phe Glu Ile Ser Asn 435 440 445 Val Arg Leu Ala Arg Val Met Leu Thr Gln Phe Ala Glu Gly Arg Leu 450 455 460 Glu Asp Gln Leu Asp Lys Tyr Asp His Trp Ala Asp Arg Phe Glu Asp 465 470 475 480 Leu Pro Leu Tyr Phe Met Thr Phe His Gly Gln Gln Ser Ile Arg Thr 485 490 495 Val Ile Asp Thr Met Gln His Ala Val Tyr Val Tyr Asp Ile Cys His 500 505 510 Val Ile Ile Asp Asn Leu Gln Phe Met Met Gly His Glu Gln Leu Ser 515 520 525 Thr Asp Arg Ile Ala Ala Gln Asp Tyr Ile Ile Gly Val Phe Arg Lys 530 535 540 Phe Ala Thr Asp Asn Asn Cys His Val Thr Leu Val Ile His Pro Arg 545 550 555 560 Lys Glu Asp Asp Asp Lys Glu Leu Gln Thr Ala Ser Ile Phe Gly Ser 565 570 575 Ala Lys Ala Ser Gln Glu Ala Asp Asn Val Leu Ile Leu Gln Asp Arg 580 585 590 Lys Leu Val Thr Gly Pro Gly Lys Arg Tyr Leu Gln Val Ser Lys Asn 595 600 605 Arg Phe Asp Gly Asp Val Gly Val Phe Pro Leu Glu Phe Asn Lys Asn 610 615 620 Ser Leu Thr Phe Ser Ile Pro Lys Asn Lys Ala Arg Leu Lys Lys 625 630 635 640 Ile Lys Asp Asp Thr Gly Pro Val Ala Lys Lys Pro Ser Ser Gly Lys 645 650 655 Lys Gly Ala Thr Thr Gln Asn Ser Glu Ile Cys Ser Gly Gln Ala Pro 660 665 670 Thr Pro Asp Gln Pro Asp Thr Ser Lys Arg Ser Lys 675 680 < 210> 4 <211> 2055 <212> DNA <213> Homo sapiens <400> 4 atgtgggtcc tcctccgaag tgggtacccc ctccgtatct tgttacccct gcgtggggag 60 tggatgggtc ggaggggcct gccccgaaac ttggccccag gccctcctcg cagacgttac 120 aggaaggaga ctctccaagc cttggatatg ccagtgttgc ctgtaactgc aactgaaatc 180 cgccagtatt tgcgggggca tgggatcccc ttccaggatg gtcacagttg cctgcgggca 240 ctgagcccct ttgcagagtc ttcacagctc aaaggccaga ctggtgttac cacttccttc 300 agcctc ttca ttgacaagac cacaggccac tttctctgca tgaccagcct agcagaaggg 360 agctgggaag acttccaggc cagcgtggag gggcgagggg atggggccag ggaggggttt 420 ctgcttagca aggcaccaga atttgaggac agcgaggagg tccggaggat ctggaaccga 480 gcaatacctc tctgggagct gcctgatcag gaggaggttc agctggctga tacaatgttt 540 ggccttacca aggttacaga tgacacactc aagcgtttca gtgtgcgata tctgcgacct 600 gctcgcagtc ttgtcttccc ttggttctcc cctgggggct caggattacg aggcctgaag 660 ctcctagagg ctaaatgcca gggggatgga gtgagctacg aggaaaccac tattccccga 720 cccagcgcct accacaatct gtttggatta ccactgatta gtcgtcgaga tgctgaggtg 780 gtactgacga gtcgtgagct tgacagcctg gccttgaacc agtccacggg gctgcctacc 840 cttactctac cccgaggaac gacctgctta ccccctgcct tactccctta cctggaacag 900 ttccggcgga ttgtattctg gttgggggat gaccttcggt cctgggaagc cgccaagttg 960 tttgcacgaa aactgaaccc caaacgatgc ttcttggtgc gaccaggaga ccagcaaccc 1020 cgtcccctgg aggccctgaa cggaggcttc aatctttctc gtattcttcg taccgccctg 1080 cctgcctggc acaagtccat cgtatctttc cggcagcttc gggaggaggt gctaggagaa 1140 ctgtcaaatg tggagcaagc a gctggcctc cgctggagcc gctttccaga cctcaatcgt 1200 atcttgaagg gacatcgaaa gggcgagctg acggtcttca cagggccaac aggcagtgga 1260 aagacgacat tcatcagtga gtatgccctg gatttgtgtt cccagggggt gaacacactg 1320 tggggtagct tcgagatcag caatgtgaga ctagcccggg tcatgctgac acagtttgcc 1380 gaggggcggc tggaagatca actggacaaa tatgatcact gggctgaccg ctttgaggac 1440 ctgcccctct atttcatgac tttccatgga cagcaaagca tcaggactgt aatagataca 1500 atgcaacatg cagtctacgt ctatgacatt tgtcatgtga tcatcgacaa cctgcagttc 1560 atgatgggtc acgagcagct gtccacagac aggatcgcag ctcaagacta catcatcggg 1620 gtctttcgga agtttgcaac agacaataac tgccatgtga cactggtcat tcacccccgg 1680 aaagaggatg atgacaagga actgcagaca gcgtccattt ttggctcagc caaagcaagc 1740 caggaagcag acaatgttct gatcctgcag gacaggaagc tggtaaccgg gccagggaaa 1800 cggtatctgc aggtgtccaa gaaccgcttt gatggagatg taggtgtctt cccgcttgag 1860 ttcaacaaga actccctcac cttctccatt ccaccaaaga acaaggcccg gctcaagaag 1920 atcaaggatg acactggacc agtggccaaa aagccctctt ctggcaaaaa gggggctacg 1980 acacagaact ctgagatttg ctcaggc cag gcccccactc ccgaccagcc agacacctcc 2040 aagcgttcaa agtga 2055 <210> 5 <211> 283 <212> PRT <213> Homo sapiens <400> 5 Met Asn Trp Thr Gly Leu Tyr Thr Leu Leu Ser Gly Val Asn Arg His 1 5 10 15 Ser Thr Ala Ile Gly Arg Val Trp Leu Ser Val Ile Phe Ile Phe Arg 20 25 30 Ile Met Val Leu Val Val Ala Ala Glu Ser Val Trp Gly Asp Glu Lys 35 40 45 Ser Ser Phe Ile Cys Asn Thr Leu Gln Pro Gly Cys Asn Ser Val Cys 50 55 60 Tyr Asp Gln Phe Phe Pro Ile Ser His Val Arg Leu Trp Ser Leu Gln 65 70 75 80 Leu Ile Leu Val Ser Thr Pro Ala Leu Leu Val Ala Met His Val Ala 85 90 95 His Gln Gln His Ile Glu Lys Lys Met Leu Arg Leu Glu Gly His Gly 100 105 110 Asp Pro Leu His Leu Glu Glu Val Lys Arg His Lys Val His Ile Ser 115 120 125 Gly Thr Leu Trp Trp Thr Tyr Val Ile Ser Val Val Phe Arg Leu Leu 130 135 140 Phe Glu Ala Val Phe Met Tyr Val Phe Tyr Leu Leu Tyr Pro Gly Tyr 145 150 155 160 Ala Met Val Arg Leu Val Lys Cys Asp Val Tyr Pro Cys Pro Asn Thr 165 170 175 Val Asp Cys Phe Val Ser Arg Pro Thr Glu Lys Thr Val Phe Thr Val 180 185 190 Phe Met Leu Ala Ala Ser Gly Ile Cys Ile Ile Leu Asn Val Ala Glu 195 200 205 Val Val Tyr Leu Ile Ile Arg Ala Cys Ala Arg Arg Ala Gln Arg Arg 210 215 220 Ser Asn Pro Pro Ser Arg Lys Gly Ser Gly Phe Gly His Arg Leu Ser 225 230 235 240 Pro Glu Tyr Lys Gln Asn Glu Ile Asn Lys Leu Leu Ser Glu Gln Asp 245 250 255 Gly Ser Leu Lys Asp Ile Leu Arg Arg Ser Pro Gly Thr Gly Ala Gly 260 265 270 Leu Ala Glu Lys Ser Asp Arg Cys Ser Ala Cys 275 280 <210> 6 <211> 852 <212> DNA <213> Homo sapiens <400> 6 atgaactgga caggtttgta caccttgctc agtggcgtga accggcattc tactgccatt 60 ggccgagtat ggctctcggt catcttcatc ttcagaatca tggtgctggt ggtggctgca 120 gagagtgtgt ggggtgatga gaaatcttcc ttcatctgca acacactcca gcctggctgc 180 aacagcgttt gctatgacca attcttcccc atctcccatg tgcggctgtg gtccctgcag 240 ctcatcctag tttccacccc agctctcctc gtggc catgc acgtggctca ccagcaacac 300 atagagaaga aaatgctacg gcttgagggc catggggacc ccctacacct ggaggaggtg 360 aagaggcaca aggtccacat ctcagggaca ctgtggtgga cctatgtcat cagcgtggtg 420 ttccggctgt tgtttgaggc cgtcttcatg tatgtctttt atctgctcta ccctggctat 480 gccatggtgc ggctggtcaa gtgcgacgtc tacccctgcc ccaacacagt ggactgcttc 540 gtgtcccgcc ccaccgagaa aaccgtcttc accgtcttca tgctagctgc ctctggcatc 600 tgcatcatcc tcaatgtggc cgaggtggtg tacctcatca tccgggcctg tgcccgccga 660 gcccagcgcc gctccaatcc accttcccgc aagggctcgg gcttcggcca ccgcctctca 720 cctgaataca agcagaatga gatcaacaag ctgctgagtg agcaggatgg ctccctgaaa 780 gacatactgc gccgcagccc tggcaccggg gctgggctgg ctgaaaagag cgaccgctgc 840 tcggcctgct ga 852 <210> 7 <211> 1956 <212> PRT <213> Homo sapiens <400> 7 Met Glu Phe Pro Ile Gly Ser Leu Glu Thr Asn Asn Phe Arg Arg Phe 1 5 10 15 Thr Pro Glu Ser Leu Val Glu Ile Glu Lys Gln Ile Ala Ala Lys Gln 20 25 30 Gly Thr Lys Lys Ala Arg Glu Lys His Arg Glu Gln Lys Asp Gln Glu 35 40 45 Glu Lys Pro Arg Pro Gln Leu As p Leu Lys Ala Cys Asn Gln Leu Pro 50 55 60 Lys Phe Tyr Gly Glu Leu Pro Ala Glu Leu Ile Gly Glu Pro Leu Glu 65 70 75 80 Asp Leu Asp Pro Phe Tyr Ser Thr His Arg Thr Phe Met Val Leu Asn 85 90 95 Lys Gly Arg Thr Ile Ser Arg Phe Ser Ala Thr Arg Ala Leu Trp Leu 100 105 110 Phe Ser Pro Phe Asn Leu Ile Arg Arg Thr Ala Ile Lys Val Ser Val 115 120 125 His Ser Trp Phe Ser Leu Phe Ile Thr Val Thr Ile Leu Val Asn Cys 130 135 140 Val Cys Met Thr Arg Thr Asp Leu Pro Glu Lys Ile Glu Tyr Val Phe 145 150 155 160 Thr Val Ile Tyr Thr Phe Glu Ala Leu Ile Lys Ile Leu Ala Arg Gly 165 170 175 Phe Cys Leu Asn Glu Phe Thr Tyr Leu Arg Asp Pro Trp Asn Trp Leu 180 185 190 Asp Phe Ser Val Ile Thr Leu Ala Tyr Val Gly Thr Ala Ile Asp Leu 195 200 205 Arg Gly Ile Ser Gly Leu Arg Thr Phe Arg Val Leu Arg Ala Leu Lys 210 215 220 Thr Val Ser Val Ile Pro Gly Leu Lys Val Ile Val Gly Ala Leu Ile 225 230 235 240 His Ser Val Lys Lys Leu Ala Asp Val Thr Ile Leu Thr Ile Phe Cys 245 250 255 Leu Ser Val Phe Ala Leu Val Gly Leu Gln Leu Phe Lys Gly Asn Leu 260 265 270 Lys Asn Lys Cys Val Lys Asn Asp Met Ala Val Asn Glu Thr Thr Asn 275 280 285 Tyr Ser Ser His Arg Lys Pro Asp Ile Tyr Ile Asn Lys Arg Gly Thr 290 295 300 Ser Asp Pro Leu Leu Cys Gly Asn Gly Ser Asp Ser Gly His Cys Pro 305 310 315 320 Asp Gly Tyr Ile Cys Leu Lys Thr Ser Asp Asn Pro Asp Phe Asn Tyr 325 330 335 Thr Ser Phe Asp Ser Phe Ala Trp Ala Phe Leu Ser Leu Phe Arg Leu 340 345 350 Met Thr Gln Asp Ser Trp Glu Arg Leu Tyr Gln Gln Thr Leu Arg Thr 355 360 365 Ser Gly Lys Ile Tyr Met Ile Phe Val Leu Val Ile Phe Leu Gly 370 375 380 Ser Phe Tyr Leu Val Asn Leu Ile Leu Ala Val Val Thr Met Ala Tyr 385 390 395 400 Glu Glu Gln Asn Gln Ala Thr Thr Asp Glu Ile Glu Ala Lys Glu Lys 405 410 415 Lys Phe Gln Glu Ala Leu Glu Met Leu Arg Lys Glu Gln Glu Val Leu 420 425 430 Ala Ala Leu Gly Ile Asp Thr Thr Leu His Ser His Asn Gly Ser 435 440 445 Pro Leu Thr Ser Lys Asn Ala Ser Glu Arg Arg His Arg Ile Lys Pro 450 455 460 Arg Val Ser Glu Gly Ser Thr Glu Asp Asn Lys Ser Pro Arg Ser Asp 465 470 475 480 Pro Tyr Asn Gln Arg Arg Met Ser Phe Leu Gly Leu Ala Ser Gly Lys 485 490 495 Arg Arg Ala Ser His Gly Ser Val Phe His Phe Arg Ser Pro Gly Arg 500 505 510 Asp Ile Ser Leu Pro Glu Gly Val Thr Asp Asp Gly Val Phe Pro Gly 515 520 525 Asp His Glu Ser His Arg Gly Ser Leu Leu Leu Gly Gly Gly Ala Gly 530 535 540 Gln Gln Gly Pro Leu Pro Arg Ser Pro Leu Pro Gln Pro Ser Asn Pro 545 550 555 560 Asp Ser Arg His Gly Glu Asp Glu His Gln Pro Pro Thr Ser Glu 565 570 575 Leu Ala Pro Gly Ala Val Asp Val Ser Ala Phe Asp Ala Gly Gln Lys 580 585 590 Lys Thr Phe Leu Ser Ala Glu Tyr Leu Asp Glu Pro Phe Arg Ala Gln 595 600 605 Arg Ala Met Ser Val Val Ser Ile Ile Thr Ser Val Leu Glu Glu Leu 610 615 620 Glu Glu Ser Glu Gln Lys Cys Pro Pro Cys Leu Thr Ser Leu Ser Gln 625 630 635 640 Lys Tyr Leu Ile Trp Asp Cys Cys Pro Met Trp Val Lys Leu Lys Thr 645 650 655 Ile Leu Phe Gly Leu Val Thr Asp Pro Phe Ala Glu Leu Thr Ile Thr 660 665 670 Leu Cys Ile Val Val Asn Thr Ile Phe Met Ala Met Glu His Gly 675 680 685 Met Ser Pro Thr Phe Glu Ala Met Leu Gln Ile Gly Asn Ile Val Phe 690 695 700 Thr Ile Phe Phe Thr Ala Glu Met Val Phe Lys Ile Ile Ala Phe Asp 705 710 715 720 Pro Tyr Tyr Tyr Phe Gln Lys Lys Trp Asn Ile Phe Asp Cys Ile Ile 725 730 735 Val Thr Val Ser Leu Leu Glu Leu Gly Val Ala Lys Lys Gly Ser Leu 740 745 750 Ser Val Leu Arg Ser Phe Arg Leu Leu Arg Val Phe Lys Leu Ala Lys 755 760 765 Ser Trp Pro Thr Leu Asn Thr Leu Ile Lys Ile Ile Gly Asn Ser Val 770 775 780 Gly Ala Leu Gly Asn Leu Thr Ile Ile Leu Ala Ile Ile Val Phe Val 785 790 795 800 Phe Ala Leu Val Gly Lys Gln Leu Leu Gly Glu Asn Tyr Arg Asn Asn 805 810 815 Arg Lys Asn Ile Ser Ala Pro His Glu Asp Trp Pro Arg Trp His Met 820 825 830 His Asp Phe Phe His Ser Phe Leu Ile Val Phe Arg Ile Leu Cys Gly 835 840 845 Glu Trp Ile Glu Asn Met Trp Ala Cys Met Glu Val Gly Gln Lys Ser 850 855 860 Ile Cys Leu Ile Leu Phe Leu Thr Val Met Val Leu Gly Asn Leu Val 865 870 875 880 Val Leu Asn Leu Phe Ile Ala Leu Leu Leu Asn Ser Phe Ser Ala Asp 885 890 895 Asn Leu Thr Ala Pro Glu Asp Asp Gly Glu Val Asn Asn Leu Gln Val 900 905 910 Ala Leu Ala Arg Ile Gln Val Phe Gly His Arg Thr Lys Gln Ala Leu 915 920 925 Cys Ser Phe Phe Ser Arg Ser Cys Pro Phe Pro Gln Pro Lys Ala Glu 930 935 940 Pro Glu Leu Val Val Lys Leu Pro Leu Ser Ser Ser Lys Ala Glu Asn 945 950 955 960 His Ile Ala Ala Asn Thr Ala Arg Gly Ser Ser Gly Gly Leu Gln Ala 965 970 975 Pro Arg Gly Pro Arg Asp Glu His Ser Asp Phe Ile Ala Asn Pro Thr 980 985 990 Val Trp Val Ser Val Pro Ile Ala Glu Gly Glu Ser Asp Leu Asp Asp 995 1000 1005 Leu Glu Asp Asp Gly Gly Glu Asp Ala Gln Ser Phe Gln Gln Glu Val 1010 1015 1020 Ile Pro Lys Gly Gln Gln Glu Gln Leu Gln Gln Val Glu Arg Cys Gly 1025 1030 1035 1040 Asp His Leu Thr Pro Arg Ser Pro Gly Thr Gly Thr Ser Ser Glu Asp 1045 1050 1055 Leu Ala Pro Ser Leu Gly Glu Thr Trp Lys Asp Glu Ser Val Pro Gln 1060 1065 1070 Val Pro Ala Glu Gly Val Asp Asp Thr Ser Ser Ser Glu Gly Ser Thr 1075 1080 1085 Val Asp Cys Leu Asp Pro Glu Glu Ile Leu Arg Lys Ile Pro Glu Le u 1090 1095 1100 Ala Asp Asp Leu Glu Glu Pro Asp Asp Cys Phe Thr Glu Gly Cys Ile 1105 1110 1115 1120 Arg His Cys Pro Cys Cys Lys Leu Asp Thr Thr Lys Ser Pro Trp Asp 1125 1130 1135 1135 Val Gly Trp Gln Val Arg Lys Thr Cys Tyr Arg Ile Val Glu His Ser 1140 1145 1150 Trp Phe Glu Ser Phe Ile Ile Phe Met Ile Leu Leu Ser Ser Gly Ser 1155 1160 1165 Leu Ala Phe Glu Asp Tyr Tyr Leu Asp Gln Lys Pro Thr Val Lys Ala 1170 1175 1180 Leu Leu Glu Tyr Thr Asp Arg Val Phe Thr Phe Ile Phe Val Phe Glu 1185 1190 1195 1200 Met Leu Leu Lys Trp Val Ala Tyr Gly Phe Lys Lys Tyr Phe Thr Asn 1205 1210 1215 Ala Trp Cys Trp Leu Asp Phe Leu Ile Val Asn Ile Ser Leu Ile Ser 1220 1225 1230 Leu Thr Ala Lys Ile Leu Glu Tyr Ser Glu Val Ala Pro Ile Lys Ala 1235 1240 1245 Leu Arg Thr Leu Arg Ala Leu Arg Pro Leu Arg Ala Leu Ser Arg Phe 1250 1255 1260 Glu Gly Met Arg Val Val Val Asp Ala Leu Val Gly Ala Ile Pro Ser 1265 1270 1275 1280 Ile Met Asn Val Leu Leu Val Cys Leu Ile Phe Trp Leu Ile Phe Ser 1285 1290 1295 Ile Met Gly Val Asn Leu Phe Ala Gly Lys Phe Trp Arg Cys Ile Asn 1300 1305 1310 Tyr Thr Asp Gly Glu Phe Ser Leu Val Pro Leu Ser Ile Val Asn Asn 1315 1320 1325 Lys Ser Asp Cys Lys Ile Gln Asn Ser Thr Gly Ser Phe Phe Trp Val 1330 1335 1340 Asn Val Lys Val Asn Phe Asp Asn Val Ala Met Gly Tyr Leu Ala Leu 1345 1350 1355 1360 Leu Gln Val Ala Thr Phe Lys Gly Trp Met Asp Ile Met Tyr Ala Ala 1365 1370 1375 Val Asp Ser Arg Glu Val Asn Met Gln Pro Lys Trp Glu Asp Asn Val 1380 1385 1390 Tyr Met Tyr Leu Tyr Phe Val Ile Phe Ile Ile Phe Gly Gly Phe Phe 1395 1400 1405 Thr Leu Asn Leu Phe Val Gly Val Ile Ile Asp Asn Phe Asn Gln Gln 1410 1415 1420 Lys Lys Lys Leu Gly Gly Gln Asp Ile Phe Met Thr Glu Glu Gln Lys 1425 1430 1435 1440 Lys Tyr Tyr Asn Ala Met Lys Lys Leu Gly Ser Lys Lys Pro Gln Lys 1445 1450 1455 Pro Ile Pro Arg Pro Leu Asn Lys Phe Gln Gly Phe Val Phe Asp Ile 1460 1465 1470 Val Thr Arg Gln Ala Phe Asp Ile Thr Ile Met Val Leu Ile Cys Leu 1475 1480 1485 Asn Met Ile Thr Met Met Val Glu Thr Asp Asp Gln Ser Glu Glu Ly s 1490 1495 1500 Thr Lys Ile Leu Gly Lys Ile Asn Gln Phe Phe Val Ala Val Phe Thr 1505 1510 1515 1520 Gly Glu Cys Val Met Lys Met Phe Ala Leu Arg Gln Tyr Tyr Phe Thr 1525 1530 1535 Asn Gly Trp Asn Val Phe Asp Phe Ile Val Val Val Leu Ser Ile Ala 1540 1545 1550 Ser Leu Ile Phe Ser Ala Ile Leu Lys Ser Leu Gln Ser Tyr Phe Ser 1555 1560 1565 Pro Thr Leu Phe Arg Val Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu 1570 1575 1580 Arg Leu Ile Arg Ala Ala Lys Gly Ile Arg Thr Leu Leu Leu Phe Ala Leu 1585 1590 1595 1600 Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu 1605 1610 1615 Val Met Phe Ile Tyr Ser Ile Phe Gly Met Ser Ser Phe Pro His Val 1620 1625 1630 Arg Trp Glu Ala Gly Ile Asp Asp Met Phe Asn Phe Gln Thr Phe Ala 1635 1640 1645 Asn Ser Met Leu Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp 1650 1655 1660 Gly Leu Leu Ser Pro Ile Leu Asn Thr Gly Pro Pro Tyr Cys Asp Pro 1665 1670 1675 1680 Asn Leu Pro Asn Ser Asn Gly Thr Arg Gly Asp Cys Gly Ser Pro Ala 1685 1690 1695 Val Gly Ile Ile Phe Phe Thr Tyr Ile Ile Ile Ser Phe Leu Ile 1700 1705 1710 Val Val Asn Met Tyr Ile Ala Val Ile Leu Glu Asn Phe Asn Val Ala 1715 1720 1725 Thr Glu Glu Ser Thr Glu Pro Leu Ser Glu Asp Asp Phe Asp Met Phe 1730 1735 1740 Tyr Glu Thr Trp Glu Lys Phe Asp Pro Glu Ala Thr Gln Phe Ile Thr 1745 1750 1755 1760 Phe Ser Ala Leu Ser Asp Phe Ala Asp Thr Leu Ser Gly Pro Leu Arg 1765 1770 1775 Ile Pro Lys Pro Asn Arg Asn Ile Leu Ile Gln Met Asp Leu Pro Leu 1780 1785 1790 Val Pro Gly Asp Lys Ile His Cys Leu Asp Ile Leu Phe Ala Phe Thr 1795 1800 1805 Lys Asn Val Leu Gly Glu Ser Gly Glu Leu Asp Ser Leu Lys Ala Asn 1810 1815 1820 Met Glu Glu Lys Phe Met Ala Thr Asn Leu Ser Lys Ser Ser Tyr Glu 1825 1830 1835 1840 Pro Ile Ala Thr Thr Leu Arg Trp Lys Gln Glu Asp Ile Ser Ala Thr 1845 1850 1855 Val Ile Gln Lys Ala Tyr Arg Ser Tyr Val Leu His Arg Ser Met Ala 1860 1865 1870 Leu Ser Asn Thr Pro Cys Val Pro Arg Ala Glu Glu Glu Ala Ala Ser 1875 1880 1885 Leu Pro Asp Glu Gly Phe Val Ala Phe Thr Ala Asn Glu Asn Cys Va l 1890 1895 1900 Leu Pro Asp Lys Ser Glu Thr Ala Ser Ala Thr Ser Phe Pro Pro Ser 1905 1910 1915 1920 Tyr Glu Ser Val Thr Arg Gly Leu Ser Asp Arg Val Asn Met Arg Thr 1925 1930 1935 Ser Ser Ser Ile Gln Asn Glu Asp Glu Ala Thr Ser Met Glu Leu Ile 1940 1945 1950 Ala Pro Gly Pro 1955 <210> 8 <211> 5871 <212> DNA <213> Homo sapiens <400> 8 atggaattcc ccattggatc cctcgaaact aacaacttcc gtcgctttac tccggagtagaga 60 ctggt cagagagaag 120 catagggagc agaaggacca agaagagaag cctcggcccc agctggactt gaaagcctgc 180 aaccagctgc ccaagttcta tggtgagctc ccagcagaac tgatcgggga gcccctggag 240 gatctagatc cgttctacag cacacaccgg acatttatgg tgctgaacaa agggaggacc 300 atttcccggt ttagtgccac tcgggccctg tggctattca gtcctttcaa cctgatcaga 360 agaacggcca tcaaagtgtc tgtccactcg tggttcagtt tatttattac ggtcactatt 420 ttggttaatt gtgtgtgcat gacccgaact gaccttccag agaaaattga atatgtcttc 480 actgtcattt acacctttga agccttgata aagatactgg caagaggatt ttgtctaaat 540 gagttcacgt acctgagaga tccttggaac tg gctggatt ttagcgtcat taccctggca 600 tatgttggca cagcaataga tctccgtggg atctcaggcc tgcggacatt cagagttctt 660 agagcattaa aaacagtttc tgtgatccca ggcctgaagg tcattgtggg ggccctgatt 720 cactcagtga agaaactggc tgatgtgacc atcctcacca tcttctgcct aagtgttttt 780 gccttggtgg ggctgcaact cttcaagggc aacctcaaaa ataaatgtgt caagaatgac 840 atggctgtca atgagacaac caactactca tctcacagaa aaccagatat ctacataaat 900 aagcgaggca cttctgaccc cttactgtgt ggcaatggat ctgactcagg ccactgccct 960 gatggttata tctgccttaa aacttctgac aacccggatt ttaactacac cagctttgat 1020 tcctttgctt gggctttcct ctcactgttc cgcctcatga cacaggattc ctgggaacgc 1080 ctctaccagc agaccctgag gacttctggg aaaatctata tgatcttttt tgtgctcgta 1140 atcttcctgg gatctttcta cctggtcaac ttgatcttgg ctgtagtcac catggcgtat 1200 gaggagcaga accaggcaac cactgatgaa attgaagcaa aggagaagaa gttccaggag 1260 gccctcgaga tgctccggaa ggagcaggag gtgctagcag cactagggat tgacacaacc 1320 tctctccact cccacaatgg atcaccttta acctccaaaa atgccagtga gagaaggcat 1380 agaataaagc caagagtgtc agagggctcc acagaagaca acaa atcacc ccgctctgat 1440 ccttacaacc agcgcaggat gtcttttcta ggcctcgcct ctggaaaacg ccgggctagt 1500 catggcagtg tgttccattt ccggtcccct ggccgagata tctcactccc tgagggagtc 1560 acagatgatg gagtctttcc tggagaccac gaaagccatc ggggctctct gctgctgggt 1620 gggggtgctg gccagcaagg ccccctccct agaagccctc ttcctcaacc cagcaaccct 1680 gactccaggc atggagaaga tgaacaccaa ccgccgccca ctagtgagct tgcccctgga 1740 gctgtcgatg tctcggcatt cgatgcagga caaaagaaga ctttcttgtc agcagaatac 1800 ttagatgaac ctttccgggc ccaaagggca atgagtgttg tcagtatcat aacctccgtc 1860 cttgaggaac tcgaggagtc tgaacagaag tgcccaccct gcttgaccag cttgtctcag 1920 aagtatctga tctgggattg ctgccccatg tgggtgaagc tcaagacaat tctctttggg 1980 cttgtgacgg atccctttgc agagctcacc atcaccttgt gcatcgtggt gaacaccatc 2040 ttcatggcca tggagcacca tggcatgagc cctaccttcg aagccatgct ccagataggc 2100 aacatcgtct ttaccatatt ttttactgct gaaatggtct tcaaaatcat tgccttcgac 2160 ccatactatt atttccagaa gaagtggaat atctttgact gcatcatcgt cactgtgagt 2220 ctgctagagc tgggcgtggc caagaaggga agcctgtctg tgctgcggag cttccgcttg 2280 ctgcgcgtat tcaagctggc caaatcctgg cccaccttaa acacactcat caagatcatc 2340 ggaaactcag tgggggcact ggggaacctc accatcatcc tggccatcat tgtctttgtc 2400 tttgctctgg ttggcaagca gctcctaggg gaaaactacc gtaacaaccg aaaaaatatc 2460 tccgcgcccc atgaagactg gccccgctgg cacatgcacg acttcttcca ctctttcctc 2520 attgtcttcc gtatcctctg tggagagtgg attgagaaca tgtgggcctg catggaagtt 2580 ggccaaaaat ccatatgcct catccttttc ttgacggtga tggtgctagg gaacctggtg 2640 gtgcttaacc tgttcatcgc cctgctattg aactctttca gtgctgacaa cctcacagcc 2700 ccggaggacg atggggaggt gaacaacctg caggtggccc tggcacggat ccaggtcttt 2760 ggccatcgta ccaaacaggc tctttgcagc ttcttcagca ggtcctgccc attcccccag 2820 cccaaggcag agcctgagct ggtggtgaaa ctcccactct ccagctccaa ggctgagaac 2880 cacattgctg ccaacactgc cagggggagc tctggagggc tccaagctcc cagaggcccc 2940 agggatgagc acagtgactt catcgctaat ccgactgtgt gggtctctgt gcccattgct 3000 gagggtgaat ctgatcttga tgacttggag gatgatggtg gggaagatgc tcagagcttc 3060 cagcaggaag tgatccccaa aggacagcag gagcagctgc agcaagtcga gaggtgtggg 3120 gaccacctga cacccaggag cccaggcact ggaacatctt ctgaggacct ggctccatcc 3180 ctgggtgaga cgtggaaaga tgagtctgtt cctcaggtcc ctgctgaggg agtggacgac 3240 acaagctcct ctgagggcag cacggtggac tgcctagatc ctgaggaaat cctgaggaag 3300 atccctgagc tggcagatga cctggaagaa ccagatgact gcttcacaga aggatgcatt 3360 cgccac tgtc cctgctgcaa actggatacc accaagagtc catgggatgt gggctggcag 3420 gtgcgcaaga cttgctaccg tatcgtggag cacagctggt ttgagagctt catcatcttc 3480 atgatcctgc tcagcagtgg atctctggcc tttgaagact attacctgga ccagaagccc 3540 acggtgaaag ctttgctgga gtacactgac agggtcttca cctttatctt tgtgttcgag 3600 atgctgctta agtgggtggc ctatggcttc aaaaagtact tcaccaatgc ctggtgctgg 3660 ctggacttcc tcattgtgaa tatctcactg ataagtctca cagcgaagat tctggaatat 3720 tctgaagtgg ctcccatcaa agcccttcga acccttcgcg ctctgcggcc actgcgggct 3780 ctttctcgat ttgaaggcat gcgggtggtg gtggatgccc tggtgggcgc catcccatcc 3840 atcatgaatg tcctcctcgt ctgcctcatc ttctggctca tcttcagcat catgggtgtg 3900 aacctcttcg cagggaagtt ttggaggtgc atcaactata ccgatggaga gttttccctt 3960 gtacctttgt cgattgtgaa taacaagtct gactgcaaga ttcaaaactc cactggcagc 4020 ttcttctggg tcaatgtgaa agtcaacttt gataatgttg caatgggtta ccttgcactt 4080 ctgcaggtgg caacctttaa aggctggatg gacattatgt atgcagctgt tgattcccgg 4140 gaggtcaaca tgcaacccaa gtgggaggac aacgtgtaca tgtatttgta ctttgtcatc 4200 ttcatcattt t tggaggctt cttcacactg aatctctttg ttggggtcat aattgacaac 4260 ttcaatcaac agaaaaaaaa gttagggggc caggacatct tcatgacaga ggagcagaag 4320 aaatactaca atgccatgaa gaagttgggc tccaagaagc cccagaagcc catcccacgg 4380 cccctgaaca agttccaggg ttttgtcttt gacatcgtga ccagacaagc ttttgacatc 4440 accatcatgg tcctcatctg cctcaacatg atcaccatga tggtggagac tgatgaccaa 4500 agtgaagaaa agacgaaaat tctgggcaaa atcaaccagt tctttgtggc cgtcttcaca 4560 ggcgaatgtg tcatgaagat gttcgctttg aggcagtact acttcacaaa tggctggaat 4620 gtgtttgact tcattgtggt ggttctctcc attgcgagcc tgattttttc tgcaattctt 4680 aagtcacttc aaagttactt ctccccaacg ctcttcagag tcatccgcct ggcccgaatt 4740 ggccgcatcc tcagactgat ccgagcggcc aaggggatcc gcacactgct ctttgccctc 4800 atgatgtccc tgcctgccct cttcaacatc gggctgttgc tattccttgt catgttcatc 4860 tactctatct tcggtatgtc cagctttccc catgtgaggt gggaggctgg catcgacgac 4920 atgttcaact tccagacctt cgccaacagc atgctgtgcc tcttccagat taccacgtcg 4980 gccggctggg atggcctcct cagccccatc ctcaacacag ggccccccta ctgtgacccc 5040 aatctgccca acagcaa tgg caccagaggg gactgtggga gcccagccgt aggcatcatc 5100 ttcttcacca cctacatcat catctccttc ctcatcgtgg tcaacatgta cattgcagtg 5160 attctggaga acttcaatgt ggccacggag gagagcactg agcccctgag tgaggacgac 5220 tttgacatgt tctatgagac ctgggagaag tttgacccag aggccactca gtttattacc 5280 ttttctgctc tctcggactt tgcagacact ctctctggtc ccctgagaat cccaaaaccc 5340 aatcgaaata tactgatcca gatggacctg cctttggtcc ctggagataa gatccactgc 5400 ttggacatcc tttttgcttt caccaagaat gtcctaggag aatccgggga gttggattct 5460 ctgaaggcaa atatggagga gaagtttatg gcaactaatc tttcaaaatc atcctatgaa 5520 ccaatagcaa ccactctccg atggaagcaa gaagacattt cagccactgt cattcaaaag 5580 gcctatcgga gctatgtgct gcaccgctcc atggcactct ctaacacccc atgtgtgccc 5640 agagctgagg aggaggctgc atcactccca gatgaaggtt ttgttgcatt cacagcaaat 5700 gaaaattgtg tactcccaga caaatctgaa actgcttctg ccacatcatt cccaccgtcc 5760 tatgagagtg tcactagagg ccttagtgat agagtcaaca tgaggacatc tagctcaata 5820caaaatgaag atgaagccac cagtatggag ctgattgccc ctgggcccta 5871 g

Claims (20)

A glycyl-tRNA synthetase (GARS) protein variant in which proline, which is the 724th amino acid residue in the amino acid sequence of SEQ ID NO: 1, is substituted with histidine. A polynucleotide encoding the GARS protein variant of claim 1. The polynucleotide of claim 2, wherein the polynucleotide comprises a nucleotide sequence in which cytosine (C), which is the 2171th base in the nucleotide sequence of SEQ ID NO: 2, is substituted with adenine (A). A protein consisting of an amino acid sequence in which proline, which is the 724th amino acid residue in the amino acid sequence of SEQ ID NO: 1, is substituted with histidine, or
Including an agent capable of specifically detecting a polynucleotide comprising a nucleotide sequence in which cytosine (C), which is the 2171th base in the nucleotide sequence of SEQ ID NO: 2, is substituted with adenine (A),
Charcot-Marie-Tooth disease (Charcot-Marie-Tooth disease) is a composition for diagnosing the onset or the risk of the onset. The method according to claim 4, wherein in the amino acid sequence of SEQ ID NO: 3, arginine at the 391th amino acid residue is substituted with histidine and the 406th amino acid residue arginine is untranslated by a stop codon.
In the nucleotide sequence of SEQ ID NO: 4, guanine (G), the 1172th base, is substituted with adenine (A), and cytosine (C), the 1216th base, is substituted with thymine (T). A composition further comprising an agent that can be detected as The amino acid sequence of claim 4, wherein in the amino acid sequence of SEQ ID NO: 5, threonine, the 18th amino acid residue, is substituted with isoleucine, the amino acid sequence in which valine, the 38th amino acid residue, is substituted with leucine in the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: The amino acid sequence in which alanine, the 88th amino acid residue, in the amino acid sequence of 5 is substituted with valine, and lysine, the 124th amino acid residue in the amino acid sequence of SEQ ID NO: 5, a frameshift occurs, and the last amino acid including the lysine An amino acid sequence in which the number of amino acids up to the residue is 72, an amino acid sequence in which methionine, the 194th amino acid residue, is substituted with arginine in the amino acid sequence of SEQ ID NO: 5, methionine, the 194th amino acid residue, is substituted with threonine in the amino acid sequence of SEQ ID NO: 5 Each protein consisting of an amino acid sequence, an amino acid sequence in which arginine, the 215th amino acid residue in the amino acid sequence of SEQ ID NO: 5, is substituted with glutamine, and an amino acid sequence in which arginine, the 1250th amino acid residue, in the amino acid sequence of SEQ ID NO: 7 is substituted with glutamine Any one or more proteins selected from the group consisting of
Or a nucleotide sequence in which cytosine (C), the 53rd base in the nucleotide sequence of SEQ ID NO: 6, is substituted with thymine (T), and guanine (G), the 112th base in the nucleotide sequence of SEQ ID NO: 6, is substituted with thymine (T) Nucleotide sequence, nucleotide sequence in which cytosine (C), the 263th base in the nucleotide sequence of SEQ ID NO: 6, is substituted with thymine (T), between bases 371 and 372 in the nucleotide sequence of SEQ ID NO: 6 two adenine (AA) In this inserted nucleotide sequence, in the nucleotide sequence of SEQ ID NO: 6, thymine (T), the 581th base, is substituted with guanine (G), in the nucleotide sequence of SEQ ID NO: 6, thymine (T), the 581th base, is cytosine ( C) a nucleotide sequence substituted with a nucleotide sequence, a nucleotide sequence in which guanine (G), which is the 644th base in the nucleotide sequence of SEQ ID NO: 6, is substituted with adenine (A), and a guanine (G) which is the 3749th base in the nucleotide sequence of SEQ ID NO: 8 A composition further comprising an agent capable of specifically detecting any one or more polynucleotides selected from the group consisting of each polynucleotide comprising the nucleotide sequence substituted with the adenine (A). The composition of claim 4, wherein the agent capable of specifically detecting the protein is an antibody. The composition of claim 4, wherein the agent capable of specifically detecting the polynucleotide is a primer, a probe, or an antisense nucleic acid. The composition of claim 8 , wherein the antisense nucleic acid is the polynucleotide or a fragment thereof, or complementary thereto. A kit for diagnosing whether or not the onset of Charcot-Marie-Tooth's disease or the risk of the onset, comprising the composition of any one of claims 4 to 9. The kit according to claim 10, wherein the kit is an RT-PCR kit, a microarray chip kit, or a protein chip kit. In order to provide information for diagnosing Charcot-Marie-Tooth disease or predicting the risk of developing Charcot-Marie-Tooth disease, in a biological sample isolated from a subject,
A protein consisting of an amino acid sequence in which proline, which is the 724th amino acid residue in the amino acid sequence of SEQ ID NO: 1, is substituted with histidine, or
A method for detecting a polynucleotide comprising a nucleotide sequence in which cytosine (C), which is the 2171th base in the nucleotide sequence of SEQ ID NO: 2, is substituted with adenine (A). 13. The method of claim 12, wherein in the biological sample isolated from the subject,
A protein consisting of an amino acid sequence in which arginine, the 391 amino acid residue in the amino acid sequence of SEQ ID NO: 3, is substituted with histidine, and arginine, the 406 amino acid residue, is terminated without being translated by a stop codon, or
In the nucleotide sequence of SEQ ID NO: 4, a polynucleotide comprising a nucleotide sequence in which guanine (G), the 1172th base is substituted with adenine (A), and cytosine (C), the 1216th base, is substituted with thymine (T), is further detected how to do it. 13. The method of claim 12, wherein in the biological sample isolated from the subject,
In the amino acid sequence in which threonine, the 18th amino acid residue in the amino acid sequence of SEQ ID NO: 5, is substituted with isoleucine, the amino acid sequence in which valine, the 38th amino acid residue, is substituted with leucine in the amino acid sequence of SEQ ID NO: 5, in the amino acid sequence of SEQ ID NO: 5 The amino acid sequence in which alanine, the 88th amino acid residue, is substituted with valine, and lysine, the 124th amino acid residue in the amino acid sequence of SEQ ID NO: 5, as the starting point, a frameshift occurs, and the number of amino acids up to the last amino acid residue including the lysine is an amino acid sequence of 72, an amino acid sequence in which methionine, the 194th amino acid residue in the amino acid sequence of SEQ ID NO: 5, is substituted with arginine, an amino acid sequence in which methionine, the 194th amino acid residue, in the amino acid sequence of SEQ ID NO: 5 is substituted with threonine, SEQ ID NO: In the amino acid sequence of 5, arginine at the 215th amino acid residue is substituted with glutamine, and in the amino acid sequence of SEQ ID NO: 7, arginine at the 1250th amino acid residue is substituted with glutamine. Each protein is selected from the group consisting of any one or more proteins,
Or a nucleotide sequence in which cytosine (C), the 53rd base in the nucleotide sequence of SEQ ID NO: 6, is substituted with thymine (T), and guanine (G), the 112th base in the nucleotide sequence of SEQ ID NO: 6, is substituted with thymine (T) Nucleotide sequence, nucleotide sequence in which cytosine (C), the 263th base in the nucleotide sequence of SEQ ID NO: 6, is substituted with thymine (T), between bases 371 and 372 in the nucleotide sequence of SEQ ID NO: 6 two adenine (AA) In this inserted nucleotide sequence, in the nucleotide sequence of SEQ ID NO: 6, thymine (T), the 581th base, is substituted with guanine (G), in the nucleotide sequence of SEQ ID NO: 6, thymine (T), the 581th base, is cytosine ( C) a nucleotide sequence substituted with a nucleotide sequence, a nucleotide sequence in which guanine (G), which is the 644th base in the nucleotide sequence of SEQ ID NO: 6, is substituted with adenine (A), and a guanine (G) which is the 3749th base in the nucleotide sequence of SEQ ID NO: 8 A method for further detecting any one or more polynucleotides selected from the group consisting of each polynucleotide comprising the nucleotide sequence substituted with the adenine (A). The method according to claim 12, wherein the method detects the protein using an agent capable of specifically detecting the protein. The method according to claim 15, wherein the agent capable of specifically detecting the protein is an antibody. The method according to claim 12, wherein the method for detecting the protein is Western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation analysis, A method using an assay selected from the group consisting of complement fixation assay, FACS and protein chip. The method of claim 12 , wherein the method detects the polynucleotide using an agent capable of specifically detecting the polynucleotide. The method according to claim 18, wherein the agent capable of specifically detecting the polynucleotide is a primer, a probe, or an antisense nucleic acid. The method according to claim 12, wherein the method for detecting the polynucleotide is selected from the group consisting of reverse transcription polymerase reaction, competitive reverse transcription polymerase reaction, real-time reverse transcription polymerase reaction, RNase protection assay (RPA), Northern blotting and DNA chip. How to use .

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