KR101755530B1 - A Marker for Detecting a Neuro-Degenerative Disease and a Method for Detecting the Neuro-Degenerative Disease by Using the Same - Google Patents

Abstract

A marker for diagnosing a degenerative neurological disease disease comprising any one or more of TSP-43 (TAR-binding protein of 43 kDa) mismatch mutations of SEQ ID NOS: 1 to 3, and i) obtaining a sample from the sample; ii) confirming whether one or more of the TSP-43 (TAR-binding protein of 43 kDa) mismatch mutations of SEQ ID NOS: 1 to 3 is expressed.

Description

A Marker for Detecting a Neuro-Degenerative Disease and a Method for Detecting the Neuro-Degenerative Disease by Using the Same}

The present invention relates to a marker for diagnosing degenerative nervous system diseases and a method for diagnosing degenerative nervous system diseases using the same. More specifically, the present invention is based on the TDP-43 (TAR-binding protein of 43kDa) missense mutation and the pathological mechanism of degenerative neurological disease, specifically identified by the applicants, the TDP-43 missense of a specific sequence invented. It relates to a marker for diagnosing neurological diseases including mutations and a method for diagnosing degenerative neurological diseases using the same.

TDP-43 is an RNA/DNA binding protein involved in transcription, RNA splicing, mRNA stability, and cytoplasmic shuttling of mRNA. TDP-43 was recently found to be a major component of ubiquitinated inclusion in amyotrophic lateral sclerosis or frontotemporal lobar degeneration.

Amyotrophic axonal sclerosis is a degenerative nervous system disease showing degeneration of motor neurons in the spinal cord and cerebral cortex, leading to paralysis and death. Frontotemporal lobe degeneration induces degeneration of the frontal and temporal lobes, which is associated with a lack of executive function, social function, and cognitive function. Clinically, amyotrophic axonal sclerosis and frontotemporal lobe degeneration can be distinguished, but there are overlapping parts in the molecular state of the patient. In many patients with amyotrophic axonal sclerosis and frontotemporal lobe degeneration, TDP-43 positive inclusion in the central nervous system appeared.

TDP-43 has characteristics that are toxic to disease, such as abnormal hyperphosphorylation, ubiquitination, and production of abnormal C-terminal fragments.

TDP-43 is mainly located in the nucleus in healthy neurons, and it has been reported that it may be abnormally located in the cytoplasm and neurites and agglutination appears in the case of disease. This is due to the loss and/or function acquisition of TDP-43. It means that it can contribute to the outbreak of.

The inventors of the present application studied the effect of the TDP-43 missense mutation on differentiated cultured neurons, and the presence or absence of the TDP-43 missense mutation having a specific sequence could be a marker capable of diagnosing degenerative neurological diseases. It was confirmed that the present invention was completed.

The present invention provides a marker for diagnosing degenerative neurological diseases comprising any one or more of TDP-43 (TAR-binding protein of 43kDa) missense mutations consisting of SEQ ID NOs: 1 to 3.

In addition, the present invention

i) obtaining a sample from the specimen;

ii) It provides a method for diagnosing a degenerative nervous system disease comprising the step of checking whether any one or more of the TDP-43 (TAR-binding protein of 43kDa) missense mutations consisting of SEQ ID NOs: 1 to 3 is expressed.

Through the marker for diagnosing degenerative nervous system disease and a method for diagnosing degenerative neurological disease using the same according to the present invention, it is possible to accurately diagnose the degenerative nervous system disease and the possibility of its occurrence.

1 shows an overview of human TDP-43, which contains a nuclear position signal (amino acids 78-84), two RNA recognition motifs, a nuclear excretion signal and a glycine rich domain.
2 is a result of analysis of the expression levels of TDP-43 wild type and missense mutations in HEK 293T cells through Western blot.
3 is a photograph showing the result of transfection of GFP into nerve cells.
4 is a graph indicating the ratio of the total neurite length.
5 is an image of TDP-43-expressing neurons showing the location of flag-TDP-43 immunostained with an anti-flag antibody.
6 is an image showing TDP-43 with mNLS, used by GFP to observe the morphology of nerve cells.
7 is a graph showing the ratio of the total length of the neurite.
Figure 8 shows the reduction of TDP-43 present in the nucleus by cytoplasmic expression of TDP-43 with mNLS by immunostaining TDP-43 or endogenous TDP-43 tagged with flag with anti-flag antibody or anti-TDP43 antibody. It is an image (the circled area indicates the nucleus of the nerve cell).

Despite recent studies on TDP-43, there is a very poor understanding of how TDP-43 is related to degenerative neurological diseases to date. In addition, many studies on TDP-43 have used yeast, fruit flies, and mammalian cell lines. In many cases, TDP-43 in these cells does not properly reflect pathological characteristics in human brain cells. Moreover, to date, there has been no research on the effect of the mutant protein of TDP-43 on the structure of mammalian neurons and the mechanism of pathological action.

The inventors of the present application analyzed the effect of the mutant protein of TDP-43 found in degenerative nervous system diseases on the structure formation or maintenance process of neurons, and discovered their pathological mechanisms, so that the TDP-43 missense mutation is a degenerative nervous system disease. It was confirmed that it can be used as a diagnostic biomarker.

The biomarker for diagnosing degenerative neurological diseases according to the present invention includes any one or more of the TDP-43 missense mutations consisting of SEQ ID NOs: 1 to 3.

SEQ ID NOs: 1 to 3 are DNA sequences of TDP-43 missense mutations designated as A315T, Q331K, and M337V, respectively.

In order to find out whether each mutant protein has an effect on the structure or function of neurons, we compared the effects by expressing them in neurons. As a result, each mutant protein increased the overall length of neurons by forming abnormal branches compared to wild type.

In addition, as a result of examining them by time, it was observed that the abnormal protrusion structure causes the segmentation of the neurite and dies in the later stages. Nerve cells perform various signal transmission at the branch protrusions. The formation of abnormal branch protrusions and increase in length are likely to disturb the signals of the nerve cells. These nerves are seen in the brain or animal models of actual Alzheimer's dementia patients. Abnormal changes in cellular structure are associated with the early pathological mechanisms of neurodegenerative diseases, such as brain degenerative diseases.

Therefore, the identification of abnormal structural changes of these neurons can be said to be an important result in predicting the initial pathological process of anterior temporal dementia or ALS due to mutations. Most of the neurodegenerative diseases are found in the later stages of the disease, and the death of nerve cells is observed, so there is a limit to obtaining specificity for each disease. However, in order to treat neurodegenerative diseases, it is very important to understand the initial pathological process, which can provide clues for the treatment of neurodegenerative diseases associated with the TDP-43 mutation.

In order to find out the pathological mechanism of how the mutant protein of TDP-43 changes the structure of neurons, we investigated the location of the mutant protein in the cell. As a result, the mutant protein of TDP-43 came out of the cell's nucleus compared to the wild type. It was confirmed that the tendency to relocate to the branches of and neurons increased. Many proteins must be present at specific locations in order to play their role in the cell, so their normal location in the cell is essential to function. The mutant protein of TDP-43 can cause a lack of native function related to RNA transcription or processing of genes related to cell structure in the nucleus by shifting its location within the cell.

As a result of confirming whether the mutant protein of TDP-43 caused the loss of TDP-43 function, causing structural abnormalities in nerve cells, abnormal branch processes were formed and the length increased due to the lack of TDP-43 function. It was confirmed that it had a similar pattern to the structural change caused by the mutant protein.

Based on the above results, the inventors of the present application confirmed that the TDP-43 missense mutation can be a marker for diagnosing degenerative neurological diseases.

In the present invention, "diagnosis" means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis is to determine the likelihood or the onset of degenerative neurological diseases, such as frontotemporal dementia or Lou Gehrig's disease.

In one embodiment, the present invention comprises the steps of: i) obtaining a sample from the specimen; ii) It may be a method for diagnosing a degenerative nervous system disease comprising the step of checking whether any one or more of the TDP-43 missense mutations consisting of SEQ ID NOs: 1 to 3 is expressed.

After collecting a biological sample, for example blood, from the specimen through step i), it is determined whether any one or more of SEQ ID NOs: 1 to 3 representing the TDP-43 missense mutant DNA sequence of step ii) is present. It can be confirmed through known methods such as Polymerase Chain Reaction (PCR).

Example

Hereinafter, examples and experimental examples will be further described, but the present invention is not limited by these examples and experimental examples.

[Experiment method and conditions]

1. Plasmid

Human or mouse-encoded cDNA was amplified through RT-PCR using the primers shown in Table 1 below from the entire RNA extract of HEK293T or mouse thymus.

[Table 1]

¹ S, Sense; A, Antisense; hTDP-43, human TDP-43; mTDP-43, mouse TDP-43

To make the TDP-43 missense mutations (A315T, Q331K, M337V), recombinant PCR was performed using the primers shown in Table 2 below including each missense mutation.

[Table 2]

In order to prepare a TDP-43 structure having a mutated nuclear localization signal (mNLS), a nuclear localization signal was modified by PCR with a specific primer described in Table 1 mentioned above. For expression in animal cells, each cDNA was made by PCR using specific primers, and then cDNA was subcloned into 3xflag-CMV7.1vector (Sigma) using the HindIII-BamHI site.

2. TDP-43 siRNA generation

To make the TDP-43 siRNA expression structure, the siRNA target finder (GenScript siRNA Target Finder) program was used to select 5 parts of the siRNA for mouse TDP-43, and then the pSUPER-GFP vector (Oligoengine) using HingIII-BglII. It was subcloned into. The TDP-43 synthetic siRNA sequence for mouse TDP-43 is sense: 5'-GAGAGGAUUUGAUCAUUAA-3' (SEQ ID NO: 20), antisense; 5'-UUAAUGAU- CAAAUCCUCUC-3' (SEQ ID NO: 21) (BioneerCo., Korea).

3. Cell culture and transfection

Neurons were cultured in E17 ICR mouse embryos, and each DNA plasmid was differentiated 5 days after culture with Lipofectamine 2000 (Invitrogen, USA) or HEK293T cells with calcium phosphate reagent (Clontech USA) or according to the manufacturer's protocol. Neurons were transfected.

To down-regulate TDP-43, TDP-43 siRNA was transfected with or without siRNA-resistant human TDP-43 on day 5 after incubation. Regarding the recovery experiment, flag-human TDP-43 (or flag-TDP-43[mNLS]) was transfected with scrambled siRNA or TDP-43 siRNA on day 5 after incubation.

Regarding TDP-43 siRNA transfection, each siRNA (75-90 pmol/μl/ well) aliquot was incubated in cell culture medium using RNAi MAX formulation (Invitrogen, Carlsbad, CA, USA), followed by manufacturer Trypsin-treated neurons were added according to the protocol of.

4. Immunocytochemistry

Two days after transfection, the transfected neurons were fixed with 4% paraformaldehyde (PFA) for 10 minutes. Regarding immunostaining, the transfected neurons were permeated in 0.1% Triton X 100 for 5 minutes and then blocked with 3% bovine serum albumin (BSA) for 1 hour at room temperature. Thereafter, the cells were incubated with anti-flag (Sigma, 1:100) or anti-mouse TDP-43 antibody (Abnova, 1:100) for 1 hour and then anti-cy3 or anti-DyLight 488 secondary antibody (JacksonLab, USA ) And incubated for 1 hour at room temperature.

5. Image analysis and quantification of total neurite length and cell viability

To measure the length of axons and dendrites, anti-Tau1 (Millipore, Billerica, USA, 1:200) positive and anti-MAP2 (Millipore, Billerica, MAUSA, 1:200) positive neurites were used, respectively. At 24 hours, 48 hours and 72 hours after transfection, neuron images for morphological analysis were obtained using an LSM 510 confocal laser scanning microscope (LSM700, CarlZeiss, Oberkochen, Germany). Confocal images of transfected neurons were obtained through sequential acquisition, and each image was 5-10 images of the Z series. The image from the stack was flattened into a single image using maximum projection. To measure the total neurite length, images were obtained using a 20X objective. All neurite development processes including dendrites and axons of individual neurons were tracked. Image J analysis program, Adobe Photoshop CS3, and Metamorph image analysis were used to quantify the total neurite length. The length of more than 20 neurons in each experimental group was measured and quantified from 3 independent experiments. Statistical analysis (one way AVOVA, Student's t-test) was performed through the GraphPad Prism 5 program.

Regarding cell survival assays, neurons were transfected with either mNLS TDP-43 or siRNA and then stained with propidium iodide 72 hours after transfection. Surviving cells were then counted based on neurons that were GFP-positive and PI-negative. Statistical analysis (one way AVOVA, Tukey's post-hoc test or Student's t-test) was performed through the GraphPad Prism 5 program. In each experiment, a total number of 100-200 cells were counted for individual conditions, and then measured and quantified from 3 independent experiments.

6. Western Blot

To confirm the expression of TDP-43 (wild type or missense mutant protein), or to investigate whether flag-tagged mouse TDP-43 was knocked down by TDP-43 siRNA, anti-flag antibody (Sigma, 1:2000), anti-GFP Western blot was performed using an antibody (SantaCruz, 1:500) and an HRP-conjugated anti-mouse or anti-rabbit secondary antibody (1:10,000-20,000).

[Example 1] Effect of TDP-43 missense mutation on neurite morphology change

Although there have been many studies on the effects of TDP-43 on cytotoxicity and neurodegeneration, little is known about the early cell pathogenesis caused by disease-related missense mutant proteins.

Thus, in order to further confirm the initial cell defect caused by the TDP-43 missense mutation, each TDP-43 missense mutation (A315T) in the glycine-rich domain (Figure 1) through recombinant PCR using a specific primer containing the mutation , Q331K, M337V) was made.

The expression level of each missense mutant protein was measured in HEK 293T cells, and the results are shown in FIG. 2. Referring to Figure 2, the expression level of each mutant TDP-43 protein was similar to that of the wild type.

To confirm the effect of the TDP-43 missense mutation on the neurite morphology, the mutant or wild-type protein was expressed in differentiated neurons. As shown in FIGS. 3 and 4, after 24 hours after transfection, increased abnormal small neurites were observed in neurons expressing the missense mutation, and total neurites were measured and quantified.

Through this, it can be confirmed that the missense mutant protein increases the abnormal neuronal morphology compared to wild-type TDP-43.

[Example 2] Position abnormality due to TDP-43 missense mutant protein

Wild-type TDP-43 was basically located in the nucleus, and was marked by DAPI (4',6'-diamidine-2'-phenylindole dihydrochloride) in neurons. However, in neurons expressing the mutant protein, it was shown that TDP-43 is located in some cytoplasm and neurite together with aggregates (FIG. 5). The number of cells showing abnormal TDP-43 position from the nucleus to the cytoplasm was increased in mutant TDP-43 expressing neurons compared to wild-type TDP-43 expressing neurons.

Through this, it can be confirmed that the TDP-43 missense mutant protein exhibits a defect in the nuclear-cytoplasmic shuttle.

[Example 3] Checking the relationship between location abnormalities and cell morphological defects

Flag missense to determine whether the intracellular location of wild-type or missense mutant TDP-43 is associated with early cell morphology defects in differentiated neurons, and to compare the effects of the cytoplasmic expression of wild-type or mutant proteins in the neurite morphology. The mutant structure was transfected into differentiated neurons. The cytoplasmic expression of wild-type TDP-43 as well as the missense mutation was increased in abnormal neurites (Figs. 6 and 7), which means that the cytoplasmic expression of TDP-43 is the cause of the abnormal neurite structure.

In fact, it was confirmed that the cell viability decreased after 72 hours of transfection due to the cytoplasmic expression of TDP-43 (FIG. 8). In addition, by reducing TDP-43 in the nucleus by the cytoplasmic expression of TDP-43, it was suggested that the abnormal position of TDP-43 may affect nuclear function.

<110> Hannam University Institute for Industry-Academia Cooperation <120> A Marker for Detecting a Neuro-Degenerative Disease and a Method for Detecting the Neuro-Degenerative Disease by Using the Same <130> p130739 <160> 21 <170> KopatentIn 2.0 <210> 1 <211> 1245 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence of A315T <400> 1 atgtctgaat atattcgggt aaccgaagat gagaacgatg agcccattga aataccatcg 60 gaagacgatg ggacggtgct gctctccacg gttacagccc agtttccagg ggcgtgtggg 120 cttcgctaca ggaatccagt gtctcagtgt atgagaggtg tccggctggt agaaggaatt 180 ctgcatgccc cagatgctgg ctggggaaat ctggtgtatg ttgtcaacta tccaaaagat 240 aacaaaagaa aaatggatga gacagatgct tcatcagcag tgaaagtgaa aagagcagtc 300 cagaaaacat ccgatttaat agtgttgggt ctcccatgga aaacaaccga acaggacctg 360 aaagagtatt ttagtacctt tggagaagtt cttatggtgc aggtcaagaa agatcttaag 420 actggtcatt caaaggggtt tggctttgtt cgttttacgg aatatgaaac acaagtgaaa 480 gtaatgtcac agcgacatat gatagatgga cgatggtgtg actgcaaact tcctaattct 540 aagcaaagcc aagatgagcc tttgagaagc agaaaagtgt ttgtggggcg ctgtacagag 600 gacatgactg aggatgagct gcgggagttc ttctctcagt acggggatgt gatggatgtc 660 ttcatcccca agccattcag ggcctttgcc tttgttacat ttgcagatga tcagattgcg 720 cagtctcttt gtggagagga cttgatcatt aaaggaatca gcgttcatat atccaatgcc 780 gaacctaagc acaatagcaa tagacagtta gaaagaagtg gaagatttgg tggtaatcca 840 ggtggctttg ggaatcaggg tggatttggt aatagcagag ggggtggagc tggtttggga 900 aacaatcaag gtagtaatat gggtggtggg atgaactttg gtacgttcag cattaatcca 960 gccatgatgg ctgccgccca ggcagcacta cagagcagtt ggggtatgat gggcatgtta 1020 gccagccagc agaaccagtc aggcccatcg ggtaataacc aaaaccaagg caacatgcag 1080 agggagccaa accaggcctt cggttctgga aataactctt atagtggctc taattctggt 1140 gcagcaattg gttggggatc agcatccaat gcagggtcgg gcagtggttt taatggaggc 1200 tttggctcaa gcatggattc taagtcttct ggctggggaa tgtag 1245 <210> 2 <211> 1245 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence of Q331K <400> 2 atgtctgaat atattcgggt aaccgaagat gagaacgatg agcccattga aataccatcg 60 gaagacgatg ggacggtgct gctctccacg gttacagccc agtttccagg ggcgtgtggg 120 cttcgctaca ggaatccagt gtctcagtgt atgagaggtg tccggctggt agaaggaatt 180 ctgcatgccc cagatgctgg ctggggaaat ctggtgtatg ttgtcaacta tccaaaagat 240 aacaaaagaa aaatggatga gacagatgct tcatcagcag tgaaagtgaa aagagcagtc 300 cagaaaacat ccgatttaat agtgttgggt ctcccatgga aaacaaccga acaggacctg 360 aaagagtatt ttagtacctt tggagaagtt cttatggtgc aggtcaagaa agatcttaag 420 actggtcatt caaaggggtt tggctttgtt cgttttacgg aatatgaaac acaagtgaaa 480 gtaatgtcac agcgacatat gatagatgga cgatggtgtg actgcaaact tcctaattct 540 aagcaaagcc aagatgagcc tttgagaagc agaaaagtgt ttgtggggcg ctgtacagag 600 gacatgactg aggatgagct gcgggagttc ttctctcagt acggggatgt gatggatgtc 660 ttcatcccca agccattcag ggcctttgcc tttgttacat ttgcagatga tcagattgcg 720 cagtctcttt gtggagagga cttgatcatt aaaggaatca gcgttcatat atccaatgcc 780 gaacctaagc acaatagcaa tagacagtta gaaagaagtg gaagatttgg tggtaatcca 840 ggtggctttg ggaatcaggg tggatttggt aatagcagag ggggtggagc tggtttggga 900 aacaatcaag gtagtaatat gggtggtggg atgaactttg gtgcgttcag cattaatcca 960 gccatgatgg ctgccgccca ggcagcacta aagagcagtt ggggtatgat gggcatgtta 1020 gccagccagc agaaccagtc aggcccatcg ggtaataacc aaaaccaagg caacatgcag 1080 agggagccaa accaggcctt cggttctgga aataactctt atagtggctc taattctggt 1140 gcagcaattg gttggggatc agcatccaat gcagggtcgg gcagtggttt taatggaggc 1200 tttggctcaa gcatggattc taagtcttct ggctggggaa tgtag 1245 <210> 3 <211> 1245 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence of M337V <400> 3 atgtctgaat atattcgggt aaccgaagat gagaacgatg agcccattga aataccatcg 60 gaagacgatg ggacggtgct gctctccacg gttacagccc agtttccagg ggcgtgtggg 120 cttcgctaca ggaatccagt gtctcagtgt atgagaggtg tccggctggt agaaggaatt 180 ctgcatgccc cagatgctgg ctggggaaat ctggtgtatg ttgtcaacta tccaaaagat 240 aacaaaagaa aaatggatga gacagatgct tcatcagcag tgaaagtgaa aagagcagtc 300 cagaaaacat ccgatttaat agtgttgggt ctcccatgga aaacaaccga acaggacctg 360 aaagagtatt ttagtacctt tggagaagtt cttatggtgc aggtcaagaa agatcttaag 420 actggtcatt caaaggggtt tggctttgtt cgttttacgg aatatgaaac acaagtgaaa 480 gtaatgtcac agcgacatat gatagatgga cgatggtgtg actgcaaact tcctaattct 540 aagcaaagcc aagatgagcc tttgagaagc agaaaagtgt ttgtggggcg ctgtacagag 600 gacatgactg aggatgagct gcgggagttc ttctctcagt acggggatgt gatggatgtc 660 ttcatcccca agccattcag ggcctttgcc tttgttacat ttgcagatga tcagattgcg 720 cagtctcttt gtggagagga cttgatcatt aaaggaatca gcgttcatat atccaatgcc 780 gaacctaagc acaatagcaa tagacagtta gaaagaagtg gaagatttgg tggtaatcca 840 ggtggctttg ggaatcaggg tggatttggt aatagcagag ggggtggagc tggtttggga 900 aacaatcaag gtagtaatat gggtggtggg atgaactttg gtgcgttcag cattaatcca 960 gccatgatgg ctgccgccca ggcagcacta cagagcagtt ggggtatggt gggcatgtta 1020 gccagccagc agaaccagtc aggcccatcg ggtaataacc aaaaccaagg caacatgcag 1080 agggagccaa accaggcctt cggttctgga aataactctt atagtggctc taattctggt 1140 gcagcaattg gttggggatc agcatccaat gcagggtcgg gcagtggttt taatggaggc 1200 tttggctcaa gcatggattc taagtcttct ggctggggaa tgtag 1245 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 4 ggcctagccg gaaaagtaaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 5 caaccaccac cccactgtct 20 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 6 tccacactga acaaaccaat tt 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 7 ggcctagcgg agatttaagc 20 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 8 cccccattct aaatctacct aacc 24 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 9 ccacactgaa caaaccaatc tg 22 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 10 cgcccaagct tggcaccatg rcrgaatata ttcgg 35 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 11 cgcggatccc attccccagc cagaaga 27 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 12 aactttggta cgttcagca 19 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 13 tgctgaacgt accaaagtt 19 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 14 gcagcactaa agagcagt 18 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 15 actgctcttt agtgctgc 18 <210> 16 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 16 tatggtgggc atgtta 16 <210> 17 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 17 taacatgccc accata 16 <210> 18 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 18 tatccaaaag ataacgctgc tgctatggat gagacagat 39 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Artificial primer sequence <400> 19 atctgtctca tccatagcag cagcgttatc ttttggata 39 <210> 20 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Artificial siRNA sequence <400> 20 gagaggauuu gaucauuaa 19 <210> 21 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Artificial siRNA sequence <400> 21 uuaaugauca aauccucuc 19

Claims (2)

In order to provide information necessary for diagnosis of degenerative nervous system diseases,
i) obtaining a sample from a specimen; And
ii) confirming the increased expression of the A315T TDP-43 mutation of SEQ ID NO: 1 in the cytoplasm and neurite, and confirming that abnormal neurite formation is increased in neurons
&Lt; / RTI &gt;
2. The method according to claim 1, wherein the neurodegenerative disease is anterior temporal dementia or Lou Gehrig's disease.

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