WO2017065335A1 - Mutant park2 gene and method for diagnosing parkinson's disease using same - Google Patents

Abstract

The present invention relates to a method for diagnosing Parkinson's disease using the PARK2 mutant gene. Specifically, the PARK2 mutation inhibits the degradation of AIMP2 and SNCA proteins, thereby causing and aggravating Parkinson's disease. Therefore, the therapeutic effect can be increased by using the composition of the present invention to check whether there is a PARK2 mutation or not and the possibility of Parkinson's disease in advance. In addition, a therapeutic agent for Parkinson's disease can be developed by screening a substance capable of changing the activity of the PARK2 mutant.

Description

Mutant PARK2 Gene and Method for Diagnosing Parkinson's Disease Using the Same

The present invention relates to a composition for diagnosing Parkinson's disease and a method for diagnosing Parkinson's disease using the same.

Parkinson's disease is a neurodegenerative disorder and is accompanied by one or more of the following symptoms: slowness, stiffness, involuntary tremor and postural anxiety. Parkinson's disease is characterized by a marked loss of dopamine neurons in the dense black matter. In addition, "a person at high risk of developing Parkinson's disease" particularly means a person who does not yet have any symptoms of Parkinson's disease but has certain risk factors.

Examples of risk factors are genetic mutations (Nussbaum NEJM 348, 2003, 25). For example, the Parkin gene of chromosome 6q25.2-27 (PARK2) is associated with juvenile Parkinsonism and is frequently found in families with autosomal recessive Parkinson inheritance (Matsumine). , Am. J. Hum. Genet., 60, 1997, 588 ,; Kitada, Nature 392, 1998, 605; Abbas, Hum.Mol. Genet. 8, 1999, 567; Tassin, Am, J, Hum. Genet. , 63, 1998, 88 and Lucking, N. Engl. J. Med. 342, 2000, 1560-7). Other loci, such as PARK6 and PARK7, have also been found to be combustible and have a high incidence in families with autosomal recessive genetic Parkinsonian history (Valente, Am. J. Hum. Genet. 68, 2001, 895; van Dujin, Am. J. Hum. Genet. 69, 2001, 629). Mutations in the alpha-synuclein gene (PARK1) have also been found in families with combustible, autosomal recessive genetic Parkinsonian history (Polymeropoulos, Science 276, 1997, 2045). In addition to genetic predisposition, environmental factors such as overexposure to pesticides (Vanacore, Neurol Sci., Sep; 23 Suppl 2, 2002, page 119) may also be representative risk factors. In addition, various stimuli such as excessive reactive oxygen species production, oxidative stress, misfolded proteins and impaired mitochondrial function are known to cause Parkinson's disease.

In addition, mutations of PARK2 (PARKIN / PAKN) have been reported recently as a cause of Parkinson's disease. However, it is not clear about the site of the mutation and its mechanism. In order to accurately diagnose Parkinson's disease, as many genetic mutations as possible should be identified, and the functions of the proteins related to the mechanisms causing Parkinson's disease and the genes that control them must also be identified.

Accordingly, the present inventors completed the present invention by finding a mutant gene that regulates a protein associated with the induction of Parkinson's disease while studying the type and mechanism of PARK2 mutation.

An object of the present invention is to provide a composition that can be used to provide information for diagnosing and diagnosing a disease of Parkinson's disease. Another object of the present invention is to provide a method for screening drugs that can treat or ameliorate Parkinson's disease.

In order to solve the above object, the present invention provides a composition for diagnosing Parkinson's disease comprising a substance capable of detecting a mutation in PARK2 of SEQ ID NO: 1.

In addition, diagnosing Parkinson's disease, comprising separating genomic DNA from a sample derived from an individual, detecting a mutation site of the PARK2 gene in the genomic DNA isolated in the step, and identifying the detected mutation site, or Methods for identifying mutation sites are provided to provide information on predicting risk of development.

In addition, a method for selecting a substance for treating and improving Parkinson's disease, comprising overexpressing PARK2 native form and mutation in a cell, adding a sample to the cell, and selecting a sample with increased activity of the PARK2 mutation. To provide.

When using the primer set of the present invention to diagnose or provide information for Parkinson's disease, genes involved in inducing Parkinson's disease can be easily detected. In addition, by overexpressing the gene and screening a substance that promotes the degradation of SNCA and AIMP2 protein of the mutated PARK2 protein, it is possible to provide a method for screening a therapeutic agent for Parkinson's disease. Through such a rapid diagnosis, the patient group that can progress to Parkinson's disease can be found in advance to provide information for proper treatment, and can provide a method for promoting treatment development.

1 shows a schematic of the PARK2 gene.

Figure 2 shows the inhibition of degradation of the matrix protein AIMP2 upon overexpression of M458L cells, confirming the loss of function of the PARK2 protein. In this case, lane 1 means MYC-AIMP2 + Mock, lane 2 means MYC-AIMP2 + PARK2-WT, and lane 3 means MYC-AIMP2 + PARK2-M458L.

Figure 3 confirms the inhibition of degradation of the matrix protein SNCA upon overexpression in M458L cells, confirming the loss of function of the PARK2 protein.

4 shows the self ubiquitination of PARK2 mutant protein.

One aspect of the present invention is to provide a composition for diagnosing Parkinson's disease, comprising a substance capable of detecting a mutation in PARK2 of SEQ ID NO: 1.

The term "Morbus Parkinson and Parkinson's disease" as used herein is used synonymously in this patent application and includes idiopathic and genetic Parkinson's disease. As used herein, the term "PARK2" refers to Parkinson's Disease Protein 2, wherein PARK2 performs the function of an E3 ubiquitin ligase enzyme in cells. The enzyme breaks down matrix proteins to maintain cell homeostasis. As a substrate of PARK2, aminoacyl-tRNA complex interacting multifuncional protein-2 (AIMP2) and SNCA (α-synuclein) are degraded by PARK2. The AIMP2 and SNCA proteins have been reported to perform neuronal cell death induction function.

Mutation in PARK2 refers to a mutation that has lost enzyme function. In particular, the mutation means that degradation of the substrate proteins AIMP2 and / or SNCA is inhibited. In addition, the mutation in the PARK2 may be that amino acids 458 in PARK2 of SEQ ID NO: 1 having an amino acid different from the natural type. The native PARK2 has 458 amino acid methionine (Met, M), but the mutant PARK2 may be other amino acid than methionine. Preferably, the 458th amino acid of mutation SEQ ID 1 in PARK2 may be leucine (Leu, L) in methionine. Preferably, the PARK2 mutation may have an amino acid sequence of SEQ ID NO.

Native PARK2 promotes the degradation of AIMP2 and / or SNCA via E3 ubiquidine such that the protein does not accumulate in cells and Parkinson's disease caused by the protein is not induced. However, mutant PARK2 inhibits the degradation of AIMP2 and / or SNCA, causing the protein to accumulate in the cell. This leads to Parkinson's disease caused by the protein.

One embodiment of the composition may include a probe, aptamer or primer that can detect a mutation of PARK2.

The term “probe” refers to a nucleic acid fragment, such as labeled RNA or DNA, that corresponds to a few bases to several tens of bases in short. 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, but is not limited thereto. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art. Probes that can recognize and bind to mutation sites can be used to recognize nucleic acids encoding PARK2 mutant proteins.

The term “primer” is a nucleic acid sequence with a short free 3 ′ hydroxyl group, which can form complementary templates and base pairs and serves as a starting point for template strand copying. Short nucleic acid sequence. By primer is meant a single-stranded oligonucleotide capable of acting as an initiation point for template-directed DNA synthesis under suitable conditions and in suitable buffers (ie, four different nucleoside triphosphates and polymerases). Suitable lengths of primers can vary depending on various factors, such as temperature and the use of the primer. In addition, the sequence of the primer need not have a sequence that is completely complementary to some sequences of the template, it is sufficient to have sufficient complementarity within the range that can hybridize with the template to perform the primer-specific action. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to the nucleotide sequence of the PARK2 mutant gene as a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to the gene sequence to act as a primer. In this case, the primer may be designed to include the mutated gene portion, or may be designed to include the mutated gene portion in the product amplified by the primer.

In one embodiment, there is provided a Parkinson's disease diagnostic kit comprising a primer set comprising a primer having a sequence of SEQ ID NO: 5 and a primer having a sequence of SEQ ID NO: 6.

Another aspect of the invention provides a kit for diagnosing Parkinson's disease.

The kit for diagnosing or predicting Parkinson's disease may include other components for making it suitable for use as a kit in addition to an agent for measuring the expression level of a PAKR2 mutant gene. For example, when the kit of the present invention is applied to a PCR amplification process, reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis) , Thermis flavus, Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors, dNTPs and primers. Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.

In addition, the material that can be included in the kit for measuring the PARK2 mutation of the present invention may be an antibody or aptamer having a specific binding site for the PARK2 mutation.

The PARK2 mutation detection kit according to the present invention can be used without limitation as long as it is for measuring the presence of PARK2 mutation in a sample. That is, the detection of the PARK2 mutation present in the kit or the sample may use conventional techniques or methods known in the art. Such methods may include, for example, radioimmunoassay (RIA), enzyme immunoassay (ELISA), and Western blotting using antibodies that specifically bind to PARK2 mutations. In addition, ELISA modifications can be used in which a large number of samples are analyzed by immobilizing a plurality of antibodies in a 96-well.

In the present invention, the kit may include substances suitable for carrying out various immunochemical reactions using the antibody, for example, a preservative of the antibody, a buffer, and the like. Further, additional reagents may be included, for example, a chromophore, a fluorescent substance, a radioisotope, or a colloid-labeled conjugate, such as a secondary antibody. The chromophore can be peroxidase, alkaline phosphatase or acid phosphatase (e.g. horseradishperoxidase) and, if it is a fluorescent substance, fluorescein carboxylic acid ( FCA), Fluorescein isothiocyanate (FITC), Fluorescein Thiourea (FTH), 7-acetoxycoumarin-3-yl, Fluorescein-5-yl, Fluorescein-6-yl, 2 ', 7'-dichlorofluorescein-5-yl, 2', 7'-dichlorofluororesin-6-yl, dihydrotetramethyllosamine-4-yl, tetramethyllomin-5-yl, tetra Methylodamin-6-yl, 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-ethyl or 4,4-difluoro- 5,7-diphenyl-4-bora-3a, 4a-diaza-s-indacene-3-ethyl is possible. In addition, the kit may include a washing solution or an eluent that can remove the substrate and the unbound protein, and the like, which will react with the enzyme and retain only the bound complex.

In the kit according to the present invention, when a sample obtained from a patient is contacted with an antibody that specifically binds to a PARK2 mutation, the sample may be diluted to an appropriate degree prior to contact with the antibody, and the antibody may be washed or separated from the complex. It may be fixed to a solid to facilitate subsequent steps. The solid phase may be glass or plastic, for example microtiter plates, rods, beads or microbeads.

 As used herein, the term “antibody” or “functional moiety thereof” is a term recognized in the art and is known immunologically (ie immunospecific to the antigen), in particular immunoglobulin molecules and immunoglobulin molecules. Molecules that contain a binding site that binds to a binding site) or an active fragment of the molecule. Immunoglobulins according to the invention can be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules . Functional portions of the antibody include, but are not limited to, single chain variable region fragments (scFv), (scFv) 2, Fab, Fab ', and F (ab') 2. The single chain variable region (scFv) refers to an antibody fragment in which a heavy chain variable region and a light chain variable region are linked through a linker peptide to take the form of a single chain polypeptide.

"Antibody" is meant to include monoclonal antibodies, polyclonal, chimeric, single chain, bispecific, apes, human and humanized antibodies, camel antibodies, diabodies, as well as functional parts or active fragments thereof. . Examples of active fragments of molecules that bind known antigens include Fab and F (ab ′) 2 fragments, including the products of Fab immunoglobulin expression libraries and epitope-binding fragments of any of the antibodies and fragments mentioned above. . Such active fragments can be derived from the antibodies of the invention by a number of techniques. For example, purified monoclonal antibodies can be cleaved with enzymes such as pepsin and subjected to HPLC gel filtration. Appropriate fractions containing Fab fragments can then be collected and concentrated by membrane filtration and the like.

An “humanized antibody” has a CDR derived from a non-human donor immunoglobulin and the engineered antibody wherein the remaining immunoglobulin-derived portion of the molecule is derived from one (or more than one) human immunoglobulin (s) Refers to one type of. Humanized antibodies may further refer to antibodies having a variable region in which one or more of its framework regions have human or primate amino acids. In addition, framework support moieties can be altered to preserve binding affinity.

The term “monoclonal antibody” is also well known in the art and refers to an antibody that is mass produced in the laboratory from a single clone and recognizes only one antigen. Monoclonal antibodies are typically prepared by fusing normally-short surviving antibody-producing B cells to fast-growing cells, such as cancer cells (or referred to as "immortal" cells). The resulting hybrid cells, or hybridomas, grow rapidly to produce clones that produce large amounts of antibodies.

The antibody can be modified by binding to various molecules such as enzymes, fluorescent materials, radioactive materials and proteins. Modified antibodies can be obtained by chemically modifying the antibody. Such modification methods are commonly used in the art.

In addition, the antibody is obtained as a chimeric antibody in which a variable region derived from a non-human antibody and a constant region derived from a human antibody are combined, or complementarity derived from a non-human antibody. It may be obtained as a humanized antibody in which a constant region is combined with a frame work region (FR) derived from a human antibody including a crystal site. Such antibodies can be prepared using methods known in the art.

Another aspect of the invention provides a method for identifying a mutation site for diagnosing a PARK2 mutation or providing information for predicting risk of onset. Specifically, the method comprises the steps of: 1) separating genomic DNA from a sample derived from an individual; 2) detecting a mutation site of the PARK2 gene in the genomic DNA isolated in step 1); And 3) identifying the mutation site detected in step 2).

In this case, the sample of step 1) is preferably blood, but is not limited thereto.

In this case, step 2) may be performed using the primers of SEQ ID NOs: 5 and 6. In addition, the mutation may be substituted with another amino acid of 458 methionine of SEQ ID NO: 1, Preferably, the 458th methionine of SEQ ID NO: 1 is preferably substituted with leucine, but is not limited thereto.

The detection in step 2) can include sequencing analysis, hybridization by microarray, allele specific PCR, dynamic allele-specific hybridization (DASH), PCR prolongation analysis or TaqMan It is preferably performed by probe PCR analysis, but is not limited thereto.

Of these methods, sequencing analysis can use conventional methods for sequencing and can be performed using an automated genetic analyzer (Sanger, F. et al., Proc. Natl. Acad. Sci. USA. , 74 (12), 5463-5467, 1977; Maxam, AM and Gilbert, W., Proc. Natl. Acad. Sci. USA., 74 (2), 560-564, 1997).

Allele-specific PCR analysis refers to a PCR method of amplifying a DNA fragment in which the mutation is located with a primer set including a primer designed with the base where the mutation is located at the 3 'end. The principle of the method is, for example, when a specific base is substituted from A to C, PCR by designing a primer containing A as the 3 'terminal base and a reverse primer capable of amplifying a DNA fragment of a suitable size. When the reaction is carried out, when the base of the mutation position is "A", the amplification reaction is normally performed, and a band of the desired position is observed. When the base is substituted with "C", the primer is complementary to the template DNA. However, the amplification reaction may not be performed properly because the 3 'end is not complementary (Newton, CR et al., Nucleic Acids Res., 17 (1), 2503-2516, 1989).

TaqMan probe PCR analysis (Livak, K. J., Genet. Anal., 14, 143-149, 1999) includes: 1) designing and constructing primers and TaqMan probes to amplify the desired DNA fragments; 2) labeling probes of different alleles with FAM dye and VIC dye (Applied Biosystems, USA); 3) performing PCR using the primers and probes using the DNA as a template; 4) after the PCR reaction is completed, analyzing and confirming the TaqMan assay plate with a sequencing analyzer; And 5) determining the genotype of the polynucleotides of step 1 from the analysis result.

Dynamic allele hybridization (DASH) analysis can be performed by a method devised by Prince et al. (Prince, J. A. et al., Genome Res. 11 (1), 152-162, 2001).

PCR extension analysis first inactivates a DNA fragment containing a base where the mutation is located by amplifying a pair of primers and then inactivating by dephosphorylation of all nucleotides added to the reaction, followed by extension primers specific for the mutation, dNTP mixtures, Didi By adding a oxynucleotide, reaction buffer and DNA polymerase to effect primer extension. At this time, the extension primer is a 3 'end of the base immediately adjacent to the 5' direction of the base where the mutation is located, the dNTP mixture excludes a nucleic acid having the same base as the didioxynucleotide, the didioxynucleotide represents a mutation It is selected from one of the base types. For example, if there is a substitution from A to C, when a mixture of dGTP, dCTP and TTP and ddATP are added to the reaction, the primer at the base where the substitution has occurred is extended by DNA polymerase and after several bases The primer extension is terminated by ddATP at the position where A base first appears. If the substitution has not occurred, since the extension reaction is terminated at the position, it is possible to determine the base type indicating the mutation by comparing the length of the extended primer.

In this case, as a detection method, when fluorescently labeling an extension primer or didioxynucleotide, the mutation may be detected by detecting fluorescence using a gene analyzer (for example, ABI's Model 3700, etc.) used for general sequencing. (Chen, J., Genome Res., 10 (4), 549-557, 2000), and matrix assisted laser desorption ionization-time of flight when using unlabeled extension primers and didioxynucleotides. The mutations can be detected by measuring molecular weight using the technique (Ross, PL, Anal. Chem, 69 (20), 4197-4202, 1997).

Another aspect of the invention provides a method for overexpressing PARK2 native and mutant intracellularly; Adding a sample to the cell; And it provides a method of selecting a substance for the treatment and improvement of Parkinson's disease comprising the step of selecting a sample with increased activity of the PARK2 mutation.

In this case, the PAKR2 mutation may be one having an amino acid sequence of SEQ ID NO: 3. In addition, increased PARK2 mutant activity can be confirmed by 1) inhibiting degradation of SNCA or AIMP2 proteins and / or 2) reducing autoubiquitination of PARK2 mutants. In addition, the sample may be any one selected from natural product extracts, compounds, nucleic acids, proteins or antibodies.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1. Cell Line Culture

Human embryonic kidney 293 (HEK293) cells, a human normal kidney cell line, were found in Dulbecco's modified Eagle's containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (DMEM). medium, Welgene, Korea) and cultured using a medium. Cell culture conditions 5% CO ₂ , 20% O ₂ , was carried out at 37 ℃ conditions.

Example 2. Plasmids

pMS1-2-PARK2, pMS1-2-PARK2-M458L, pCMV-MYC-AIMP2 and pcDNA3.1-synuclein were cloned and used (pMS1-2 s1 tag mammalian vector was obtained from Abcam, pCMV-MYC vector Clontech, and the pcDNA3.1 vector were obtained from Thermo Fisher Scientific).

Example 3. Transduction

In order to overexpress a target protein in the cell lines HEK293, HEK293 cells were dispensed to 2 × 10 ^5/60 ㎜. After 12 hours, the plasmids were transduced in single or multiple configurations as indicated in FIGS. 2-4 using transduction reagents (PEI, polyethylenimide, Polysciences, USA) after replacement with serum and antibiotic deficient media. Thereafter, further incubation for 48 hours.

Example 4. Antibodies

Anti-MYC (LF-MA0046, AbFrontier, Korea), anti-SNCA (# 610737, BD biosciences, USA), anti-PARK2 (sc-32282, Santa Cruz Biotechnology, USA), anti-GAPDH (LF-PA0212, AbFrontier ), g-Ubiquitin (sc-8017, Santa Cruz Biotechnology), anti-rabbit horseradish peroxidase (HRP) (# 31463, Thermo Fisher Scientific, USA), and anti-mouse HRP (# 31439, Thermo Fisher Scientific) antibodies Western blot was performed.

Example 5. Western Blot

Each cell was collected and then disrupted by the addition of extraction buffer (50 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.4 mM phenylmethylsulfonyl fluoride (PMSF)). After separating the supernatant by centrifugation at 13,000 rpm for 10 minutes, the concentration of the protein present in the cell lysate was quantified by the Bradford protein assay. Twenty ug of protein samples, respectively, were loaded and separated on 10% or 12% SDS-PAGE gels and then transferred to nitrocellulose membranes (GE Healthcare life sciences, USA). Thereafter, the transferred membrane was blocked with a skim milk for 30 minutes, and then the primary antibody was added and reacted at 4 ° C. for 4 hours. Thereafter, HRP-conjugated secondary antibodies were attached and confirmed by photosensitive X-ray film using an ECL solution (AbFrontier). At this time, it was confirmed that the same amount of protein was used using anti-GAPDH antibody as a quantitative control.

Figure 2:

AIMP2 plasmid, a target for degradation of PARK2 enzyme, was transduced in HEK293 cells with PARK2 WT or -M458L plasmid. After 48 hours, cells were collected and subjected to western blot with anti-MYC, anti-PARK2, anti-GAPDH antibodies. As a result, AIMP2 was degraded by PARK2 WT, but no degradation by PARK2 M458L was observed. This means that the E3 ligase function of PARK2 M458L was lost. GAPDH is a quantitative control that indicates that the same amount of protein was loaded.

Figure 3:

SNCA plasmid, a target for degradation of PARK2 enzyme, was transduced in HEK293 cells with PARK2 WT or M458L plasmid. After 48 hours, cells were collected and subjected to western blot with anti-SNCA, anti-PARK2, anti-GAPDH antibodies. As a result, SNCA was degraded by PARK2 WT, but no degradation by PARK2 M458L was observed. This means that the E3 ligase function of PARK2 M458L was lost. GAPDH is a quantitative control that indicates that the same amount of protein was loaded.

Example 6. Intracellular ( in cell) Autoubiquitination Investigation

The wild and mutated forms of PARK2 (M458L) were overexpressed in HEK293 cells, collected 48 hours later, and disrupted by addition of lysis buffer. After quantification by the Bradford method, 1 ug of anti-PARK antibody was added to 1 mg of protein sample, followed by reaction for 12 hours at 4 ° C. rotator. Thereafter, 10 μl of protein A / G bead (sc-2003, Santa Cruz Biotechnology) was added, followed by reaction at 4 ° C. for 4 hours. The immunoprecipitates were precipitated, washed three times with the extraction buffer, and the reaction was stopped by addition of the SDS indicator buffer. The samples were subjected to western blot to observe the degree of autoubiquitination of PARK2 itself.

Figure 4:

Since PARK2 enzyme can promote its ubiquitination, it was confirmed by the self-ubiquitination test whether it possesses the function. Wild-type and M458L were transduced into HEK293 cells, and cells were collected and disrupted 48 hours later. After collecting the PARK2 protein by immunoprecipitation, auto-ubiquitination was confirmed using PARK2 and Ub antibodies. (Ubiquitinated bands are attracted by covalent bonds with Ub). As a result, it was confirmed that M458L lost its self-ubiquitination function. Arrows indicate PARK2.

SEQ ID NO: 1:

MIVFVRFNSSHGFPVEVDSDTSIFQLKEVVAKRQGVPADQLRVI

FAGKELRNDWTVQNCDLDQQSIVHIVQRPWRKGQEMNATGGDDPRNAAGGCEREPQSL

TRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAGSPAGRSIYNSFYVYCKGPCQRVQPG

KLRVQCSTCRQATLTLTQGPSCWDDVLIPNRMSGECQSPHCPGTSAEFFFKCGAHPTS

DKETSVALHLIATNSRNITCITCTDVRSPVLVFQCNSRHVICLDCFHLYCVTRLNDRQ

FVHDPQLGYSLPCVAGCPNSLIKELHHFRILGEERYNRYQQYGAEECVLQMGGVLCPR

PGCGAGLLPEPDQRKVTCEGGNGLGCGFAFCRECKETYHEGECSAVFEASGTTTQAYR

VDERAAEQARWEAASKETIKKTTKPCPRCHVPVEKNGGCMHMKCPQPQCRLEWCWNCG

CEWNRVC M GDHWFDV

SEQ ID NO: 2

        1 atgatagtgt ttgtcaggtt caactccagc catggtttcc cagtggaggt cgattctgac

       61 accagcatct tccagctcaa ggaggtggtt gctaagcgac agggggttcc ggctgaccag

      121 ttgcgtgtga ttttcgcagg gaaggagctg aggaatgact ggactgtgca gaattgtgac

      181 ctggatcagc agagcattgt tcacattgtg cagagaccgt ggagaaaagg tcaagaaatg

      241 aatgcaactg gaggcgacga ccccagaaac gcggcgggag gctgtgagcg ggagccccag

      301 agcttgactc gggtggacct cagcagctca gtcctcccag gagactctgt ggggctggct

      361 gtcattctgc acactgacag caggaaggac tcaccaccag ctggaagtcc agcaggtaga

      421 tcaatctata acagctttta tgtgtattgc aaaggcccct gtcaaagagt gcagccggga

      481 aaactcaggg tacagtgcag cacctgcagg caagcaacgc tcaccttgac ccagggtcca

      541 tcttgctggg atgatgtttt aattccaaac cggatgagtg gtgaatgcca atccccacac

      601 tgccctggga ctagtgcaga atttttcttt aaatgtggag cacaccccac ctctgacaag

      661 gaaacatcag tagctttgca cctgatcgca acaaatagtc ggaacatcac ttgcattacg

      721 tgcacagacg tcaggagccc cgtcctggtt ttccagtgca actcccgcca cgtgatttgc

      781 ttagactgtt tccacttata ctgtgtgaca agactcaatg atcggcagtt tgttcacgac

      841 cctcaacttg gctactccct gccttgtgtg gctggctgtc ccaactcctt gattaaagag

      901 ctccatcact tcaggattct gggagaagag cggtacaacc ggtaccagca gtatggtgca

      961 gaggagtgtg tcctgcagat ggggggcgtg ttatgccccc gccctggctg tggagcgggg

     1021 ctgctgccgg agcctgacca gaggaaagtc acctgcgaag ggggcaatgg cctgggctgt

     1081 gggtttgcct tctgccggga atgtaaagaa acgtaccatg aaggggagtg cagtgccgta

     1141 tttgaagcct caggaacaac tactcaggcc tacagagtcg atgaaagagc cgccgagcag

     1201 gctcgttggg aagcagcctc caaagaaacc atcaagaaaa ccaccaagcc ctgtccccgc

     1261 tgccatgtac cagtggaaaa aaatggaggc tgcatgcaca tgaagtgtcc gcagccccag

1321 tgcaggctcg agtggtgctg gaactgtggc tgcgagtgga accgcgtctg c atg ggggac

     1381 cactggttcg acgtgtag

SEQ ID NO: 3:

MIVFVRFNSSHGFPVEVDSDTSIFQLKEVVAKRQGVPADQLRVI

FAGKELRNDWTVQNCDLDQQSIVHIVQRPWRKGQEMNATGGDDPRNAAGGCEREPQSL

TRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAGSPAGRSIYNSFYVYCKGPCQRVQPG

KLRVQCSTCRQATLTLTQGPSCWDDVLIPNRMSGECQSPHCPGTSAEFFFKCGAHPTS

DKETSVALHLIATNSRNITCITCTDVRSPVLVFQCNSRHVICLDCFHLYCVTRLNDRQ

FVHDPQLGYSLPCVAGCPNSLIKELHHFRILGEERYNRYQQYGAEECVLQMGGVLCPR

PGCGAGLLPEPDQRKVTCEGGNGLGCGFAFCRECKETYHEGECSAVFEASGTTTQAYR

VDERAAEQARWEAASKETIKKTTKPCPRCHVPVEKNGGCMHMKCPQPQCRLEWCWNCG

CEWNRVC L GDHWFDV

SEQ ID NO 4:

        1 atgatagtgt ttgtcaggtt caactccagc catggtttcc cagtggaggt cgattctgac

       61 accagcatct tccagctcaa ggaggtggtt gctaagcgac agggggttcc ggctgaccag

      121 ttgcgtgtga ttttcgcagg gaaggagctg aggaatgact ggactgtgca gaattgtgac

      181 ctggatcagc agagcattgt tcacattgtg cagagaccgt ggagaaaagg tcaagaaatg

      241 aatgcaactg gaggcgacga ccccagaaac gcggcgggag gctgtgagcg ggagccccag

      301 agcttgactc gggtggacct cagcagctca gtcctcccag gagactctgt ggggctggct

      361 gtcattctgc acactgacag caggaaggac tcaccaccag ctggaagtcc agcaggtaga

      421 tcaatctata acagctttta tgtgtattgc aaaggcccct gtcaaagagt gcagccggga

      481 aaactcaggg tacagtgcag cacctgcagg caagcaacgc tcaccttgac ccagggtcca

      541 tcttgctggg atgatgtttt aattccaaac cggatgagtg gtgaatgcca atccccacac

      601 tgccctggga ctagtgcaga atttttcttt aaatgtggag cacaccccac ctctgacaag

      661 gaaacatcag tagctttgca cctgatcgca acaaatagtc ggaacatcac ttgcattacg

      721 tgcacagacg tcaggagccc cgtcctggtt ttccagtgca actcccgcca cgtgatttgc

      781 ttagactgtt tccacttata ctgtgtgaca agactcaatg atcggcagtt tgttcacgac

      841 cctcaacttg gctactccct gccttgtgtg gctggctgtc ccaactcctt gattaaagag

      901 ctccatcact tcaggattct gggagaagag cggtacaacc ggtaccagca gtatggtgca

      961 gaggagtgtg tcctgcagat ggggggcgtg ttatgccccc gccctggctg tggagcgggg

     1021 ctgctgccgg agcctgacca gaggaaagtc acctgcgaag ggggcaatgg cctgggctgt

     1081 gggtttgcct tctgccggga atgtaaagaa acgtaccatg aaggggagtg cagtgccgta

     1141 tttgaagcct caggaacaac tactcaggcc tacagagtcg atgaaagagc cgccgagcag

     1201 gctcgttggg aagcagcctc caaagaaacc atcaagaaaa ccaccaagcc ctgtccccgc

     1261 tgccatgtac cagtggaaaa aaatggaggc tgcatgcaca tgaagtgtcc gcagccccag

1321 tgcaggctcg agtggtgctg gaactgtggc tgcgagtgga accgcgtctg c ctg ggggac

     1381 cactggttcg acgtgtag

SEQ ID NO: 5 (Forward primer (5 '→ 3'):

TCTAGGCTAGCGTGCTGGTT

SEQ ID NO: 6 Reverse primer (5 '→ 3'):

TGCAATTTGGCTGTAGTTGG

Claims (14)

1. Parkinson's disease diagnostic composition comprising a substance capable of detecting a mutation in PARK2 of SEQ ID NO: 1.
2. The method of claim 1,

   Wherein said mutation is the 458th amino acid of SEQ ID NO: 1 is substituted with another amino acid methionine.
3. The method of claim 1,

   Wherein said mutation is a 458th methionine of SEQ ID NO: 1 substituted with leucine.
4. The method of claim 1,

   The composition capable of detecting a mutation of PARK2 is a probe, aptamer or primer, composition.
5. The method of claim 4, wherein

   The primer comprises SEQ ID NO: 5 and SEQ ID NO: 6.
6. A primer set comprising a primer having a sequence of SEQ ID NO: 5 and a primer having a sequence of SEQ ID NO: 6; or

   A Parkinson's disease diagnostic kit comprising an antibody capable of binding a protein substituted with leucine of the 458th amino acid of the PARK2 mutation of SEQ ID NO: 1.
7.  1) separating genomic DNA from a sample derived from the individual;

    2) detecting a mutation site of the PARK2 gene in the genomic DNA isolated in step 1); And

    3) A method for identifying a mutation site for providing information of diagnosis or predicting risk of developing Parkinson's disease, comprising identifying a mutation site detected in step 2).
8. The method of claim 7, wherein

   Step 2) is to be carried out using the primers of SEQ ID NOs: 5 and 6.
9. The method of claim 7, wherein

   Wherein said mutation is that the 458th amino acid of SEQ ID NO: 1 is substituted with an amino acid other than methionine.
10. The method of claim 7, wherein

    Wherein said mutation is that the 458th methionine of SEQ ID NO: 1 is substituted with leucine.
11. 1) overexpressing PARK2 native form and mutation intracellularly;

    2) adding a sample to the cells; And

    3) selecting a sample having increased activity of the PARK2 mutation;

    How to select materials for treating and improving Parkinson's disease.
12. The method of claim 11,

    Wherein said PAKR2 mutation has the amino acid sequence of SEQ ID NO: 3.
13. The method of claim 11,

    Increase in PARK2 Mutant Activity

    1) degradation of SNCA or AIMP2 protein is inhibited and / or

    2) confirming through decreasing autoubiquitination of the PARK2 mutation.
14. The method of claim 11,

    The sample is any one selected from natural product extracts, compounds, nucleic acids, proteins or antibodies.

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