KR101866484B1 - OPA1 as a causative gene of optic atrophy or sensorimotor neuropathy and method for diagnosing the disease using the same - Google Patents

Abstract

A composition or kit for diagnosing optic atrophy or sensory motor neuropathy comprising OPA1 protein variant or OPA1 gene variant, and a preparation capable of specifically detecting them, and a method of diagnosing said disease using the same. It is possible to effectively predict or diagnose optic atrophy or sensory motor neuropathy by detecting the mutant, and further to develop a drug for treating optic atrophy or sensory motor neuropathy.

Description

[0001] The present invention relates to OPA1 as a causative gene of optic atrophy or sensory motor neuropathy, and to a method of diagnosing the disease using the same. [0002] The present invention relates to a method for diagnosing optic atrophy or sensorimotor neuropathy,

OPA1 protein variant or OPA1 gene variant, and an agent capable of specifically detecting them. The present invention relates to a composition or kit for diagnosing optic atrophy or sensory motor neuropathy.

Optic atrophy is a characteristic disorder of neuropsychiatric symptoms such as decreased visual acuity due to degeneration and disappearance of bilateral optic nerve. Patients with optic atrophy usually present with moderate visual deficits and color deficits associated with defects in the central and subcentral regions of the visual field. The severity of the disease is very diverse. Some patients have normal vision, but others have blindness. In ophthalmic examination through ophthalmoscopy, pale or atrophy of optic disc not accompanied by other findings can be confirmed, which is due to the death of retinal ganglion cells. Patients with optic atrophy of about 20% exhibit systemic symptoms such as sensory neuron loss, or less common but chronic progressive optic atrophy, myopathy, peripheral neuropathy, multiple sclerosis, rigorous bilateral paralysis, or cataract, in addition to eye symptoms.

Peripheral neuropathy is a peripheral neuropathy caused by a mutation in the causative gene and is associated with ataxic sensory neuropathy, nonataxic sensory neuropathy, and sensory motor neuropathy (sensorimotor neuropathy). Each disease is divided into several subtypes depending on the type of mutation of the gene. Sensory motor neuropathy is a subtype of sensorimotor polyneuropathy and multiple mononeuropathy. There are various subtypes, and the cause genes and clinical symptoms are slightly different for each subtype.

So far, no method to prevent or cure optic atrophy is known. On the other hand, a method for detecting a homozygous TMEM126A mutation with a marker for diagnosing non-syndromic febrile optic atrophy is known (US Patent Publication No. 2016-0032382). However, mutations that cause optic nerve atrophy are diverse and need constant research. In addition, for the development of therapeutic methods such as gene therapy and drug therapy, it is necessary to accurately diagnose the causes of optic atrophy.

Therefore, the inventors of the present invention have made efforts to investigate the causes of optic nerve atrophy and sensory motor neuropathy. As a result, they have identified novel mutant genes that have not been reported previously, and the mutant genes are useful for diagnosis of optic atrophy and sensory motor neuropathy The present invention has been completed.

U.S. Published Patent Application No. US 2016-0032382 (2016.02.04)

In one aspect, a mutation (L620fs) in which a frame shift occurs with leucine (L) at position 620 in the amino acid sequence of SEQ ID NO: 1, or a mutation in which arginine (R) at position 905 is substituted with glutamine (R905Q), or a polynucleotide encoding OPA1 (optic atrophy 1) protein variant thereof.

Another aspect is a protein comprising an amino acid sequence comprising an L620fs or R905Q mutation in the amino acid sequence of SEQ ID NO: 1 or a polynucleotide consisting of a nucleotide sequence comprising 1857-1858 delinsT or 2714G> A mutation in the nucleotide sequence of SEQ ID NO: 2 The present invention provides a composition for diagnosing the onset or the risk of the onset of optic nerve atrophy or sensory motor neuropathy.

Another aspect provides a kit for diagnosing the onset or the risk of developing optic atrophy or sensory motor neuropathy comprising the composition.

Another aspect is the use of an amino acid sequence comprising an L620fs or R905Q mutation in the amino acid sequence of SEQ ID NO: 1 in a biological sample isolated from an individual to diagnose optic atrophy or sensory motor neuropathy or to provide information for predicting the risk of onset Or a polynucleotide consisting of a nucleotide sequence comprising 1857-1858 deLinsT or 2714G> A mutation in the nucleotide sequence of SEQ ID NO: 2.

An aspect of the present invention is a mutation (L620fs) in which frame-shifted frames (L620fs) are generated starting from leucine (L) at position 620 in the amino acid sequence of SEQ ID NO: 1 or arginine (R) at position 905 is replaced with glutamine An OPA1 (optic atrophy 1) protein variant consisting of an amino acid sequence containing a substituted mutation (R905Q) or a polynucleotide encoding the same.

The OPA1 protein variant may be a separate OPA1 protein variant.

The frame shift may be such that two cytosines (C) at positions 1857 and 1858 in the nucleotide sequence of SEQ ID NO: 2 are deleted and one thymine (T) is inserted at that position.

Specifically, in the L620fs mutation, a frame shift occurs starting from Leucine (L) at position 620 in the amino acid sequence of SEQ ID NO: 1, and the thirteenth position from the 620th position is coded by the termination codon, L620fs * Can be 13 variations.

The polynucleotide encoding the OPA1 protein variant is a mutant in which two cytosines (C) at positions 1857 and 1858 are deleted and one thymine (T) is inserted at that position in the nucleotide sequence of SEQ ID NO: 2 (1857- Or a polynucleotide consisting of a nucleotide sequence including a mutation in which guanine (G) at position 2714 is substituted with adenine (A) (2714G > A).

Thus, the OPA1 protein variant is encoded by a polynucleotide consisting of a nucleotide sequence comprising the 1857-1858 delins T mutation in the nucleotide sequence of SEQ ID NO: 2 and is selected from the OPA1 protein variant consisting of the amino acid sequence comprising the L620fs mutation in the amino acid sequence of SEQ ID NO: Lt; / RTI >

In addition, the OPA1 protein variant is encoded by a polynucleotide consisting of a nucleotide sequence comprising the nucleotide sequence of 2714G > A in the nucleotide sequence of SEQ ID NO: 2, and the OPA1 protein variant comprising the amino acid sequence comprising the R905Q mutation in the amino acid sequence of SEQ ID NO: Lt; / RTI >

A polynucleotide consisting of a nucleotide sequence comprising 1857-1858 delins T or 2714G > A mutation in the nucleotide sequence of SEQ ID NO: 2 may be an OPA1 gene mutant.

OPA1 ( optic atrophy 1 ) gene is a gene encoding a protein that regulates mitochondrial fusion and cristae structure in mitochondrial inner membrane and contributes to ATP synthesis and cell death. It is registered as NCBI Accession number NM_015560.2 For example, a gene consisting of the nucleotide sequence shown in SEQ ID NO: 2.

The OPA1 protein is a protein encoded by the OPA1 gene and may be a protein consisting of the amino acid sequence shown in SEQ ID NO:

The term " OPA1 protein variant " means that a mutation has occurred in a part of the amino acid sequence of the OPA1 protein represented by SEQ ID NO: 1. Specifically, it may be a protein encoded by an OPA1 gene mutant consisting of a nucleotide sequence comprising 1857-1858 delins T or 2714G> A mutation in the nucleotide sequence of SEQ ID NO: 2, and more specifically, the amino acid sequence of SEQ ID NO: 1 L620fs, or R905Q mutations.

The term " OPA1 gene mutant " means that a mutation occurs in a part of the nucleotide sequence of the OPA1 gene represented by SEQ ID NO: 2. Specifically, made in the nucleotide sequence of SEQ ID NO: 2 as a nucleotide sequence comprising or 1857-1858delinsT 2714G> A mutation OPA1 Lt; / RTI > The cytosine (C) at position 1858 in the nucleotide sequence of SEQ ID NO: 2 and the cytosine (C) at position 1858 and the guanine (G) at position 2714 may be sequence positions that are well preserved among vertebrates .

The OPA1 Gene variants include OPA1 May be produced by frame-shift mutations of genes, or by point mutations, specifically missense mutations.

An OPA1 protein variant consisting of the amino acid sequence comprising the L620fs or R905Q mutation in the amino acid sequence of SEQ ID NO: 1 or an OPA1 gene variant consisting of the nucleotide sequence comprising the 1857-1858 delinsT or 2714G> A mutation in the nucleotide sequence of SEQ ID NO: 2 It may be a diagnostic marker of optic atrophy or sensory motor neuropathy.

Thus, the protein OPA1 L620fs or R905Q mutation region of the mutant, or 1857-1858delinsT or 2714G> A transition portion of the OPA1 gene variant may be a mutation marker for diagnosis of optic atrophy, or sensorimotor neuropathies. Specifically, a mutation of c.1857-1858delinsT OPA1 gene, or c.2714G> A mutation may be a respective recessive mutations (recessive mutation) marker. In addition, the c.1857-1858delinsT and c.2714G> A compound heterozygous mutations of the OPA1 gene may be mutation markers for the diagnosis of optic atrophy or sensory motor neuropathy, and more specifically, optic atrophy and sensory May be a mutation marker for the diagnosis of an individual suffering from a complex symptom of motor neuropathy.

Optic atrophy is a characteristic disease characterized by visual-ocular symptoms such as decreased visual acuity due to degeneration of the optic nerve. It can cause symptoms such as visual deficiency, color vision disorder, blindness, papules or papules of the optic disc. Optic atrophy can be accompanied by symptoms such as peripheral neuropathy and cataracts.

Sensorimotor neuropathy is a type of peripheral neuropathy that may present with symptoms such as ataxia, sensory disturbances, muscle weakness, and muscle atrophy.

The term " diagnosis " means identifying the presence or characteristic of a pathological condition. Therefore, the above-mentioned " diagnosis of optic atrophy or sensory motor neuropathy " may refer to whether or not the optic nerve atrophy or sensory motor neuropathy is caused or predicted, and to predict the risk of onset.

The term " diagnosis marker " may refer to a substance capable of diagnosing the onset or onset of the optic atrophy or sensory motor neuropathy.

Selection and application of significant diagnostic markers can determine the reliability of diagnostic results. Significant diagnostic markers may be markers with high reliability with high validity and consistency in repeated measurements. OPA1 made in the protein variants, or the nucleotide sequence of SEQ ID NO: 2 consisting of the amino acid sequence comprising L620fs or R905Q mutations in the amino acid sequence of SEQ ID NO: 1 as a diagnostic marker to a nucleotide sequence comprising or 1857-1858delinsT 2714G> A mutation OPA1 Gene variants are highly reliable markers that are detected only in patients with optic atrophy or sensory motor neuropathy and rarely in normal controls. Therefore, the diagnosis result based on the result obtained by detecting the presence or absence of the mutant can be reasonably reliable.

Another aspect is a protein comprising an amino acid sequence comprising an L620fs or R905Q mutation in the amino acid sequence of SEQ ID NO: 1 or a polynucleotide consisting of a nucleotide sequence comprising 1857-1858 delinsT or 2714G> A mutation in the nucleotide sequence of SEQ ID NO: 2 The present invention provides a composition for diagnosing the onset or the risk of the onset of optic nerve atrophy or sensory motor neuropathy.

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

The agent capable of specifically detecting the protein may be a substance capable of specifically detecting L620fs or R905Q mutation sites.

The term " agent capable of specifically detecting the L620fs or R905Q mutation site of a protein " means a substance that can be used to specifically identify or detect the L620fs or R905Q mutation site of the protein in a sample of an individual .

The agent capable of specifically detecting the protein may be an OPA1 protein variant, for example, a specific compound or a synthetic compound targeting a L620fs or R905Q mutation site of an OPA1 mutant, more specifically, an OPA1 protein variant Specific antibodies, and more specifically antibodies specific to OPA1 protein variants comprising the L620fs or R905Q variant of the protein of SEQ ID NO: 1, but the type thereof is not necessarily limited thereto.

The term " antibody ", as it is known in the art, refers to a specific protein molecule directed against an antigenic site. The antibody specific to the OPA1 protein variant may be obtained by cloning a gene encoding an OPA1 protein variant into an expression vector according to a conventional method to obtain an OPA1 protein variant encoded by the gene, ≪ / RTI > Also included are partial peptides that may be made from the OPA1 protein variants, and the partial peptides 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 any polyclonal antibody, monoclonal antibody or antigen-binding property thereof may be included in the antibody and all the immunoglobulin antibodies may be included. Further, the antibody may also include a special antibody such as a humanized antibody.

A polyclonal antibody can be produced by methods well known in the art for injecting an OPA1 protein variant antigen described above into an animal and obtaining blood from an animal to obtain an antibody-containing serum. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, dogs, and the like.

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

The antibody capable of detecting the OPA1 protein variant may comprise a complete form having two full-length light chains and two full-length heavy chains as well as a functional fragment of the antibody molecule. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

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

The agent capable of specifically detecting the polynucleotide can be a substance capable of specifically detecting 1857-1858 deLinsT or 2714G> A mutation site.

The term " agent capable of specifically detecting the 1857-1858 delins T or 2714G> A mutation site of a polynucleotide " specifically refers to a 1857-1858 delins T or 2714 G> A mutation site of the polynucleotide in a sample of an individual Means a substance that can be used for detection.

The agent capable of specifically detecting the polynucleotide may be a substance that specifically binds to the OPA1 gene mutant, specifically, a substance that specifically binds to the 1857-1858 deLinsT or 2714G> A mutation site of the OPA1 gene mutant Probe, or antisense nucleic acid that specifically binds to the 1857-1858 deLinsT or 2714G> A mutation site of the OPA1 gene mutant, but the type is not necessarily limited thereto.

The primer, probe, or antisense nucleic acid specifically binds to a polynucleotide consisting of a nucleotide sequence comprising 1857-1858 deLinsT or 2714G> A mutation in the nucleotide sequence of SEQ ID NO: 2, and does not specifically bind to the base sequence of other nucleic acid material It may not be.

The primer, probe, or antisense nucleic acid may be prepared by a conventional art using OPA1 Can be designed based on the nucleotide sequence of the gene variant.

The term " primer " refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a complementary template and serving as a starting point for template strand copying. Lt; RTI ID = 0.0 > 50 < / RTI > Primers are usually synthesized but can also be used in naturally occurring nucleic acids. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, but is sufficiently complementary that it can hybridize with the template. Primers are designed to initiate DNA synthesis in the presence of a polymerization reaction (i. E., A reagent for DNA polymerase or reverse transcriptase and four different nucleoside triphosphates) at the appropriate buffer solution and temperature The sense of the nucleotide sequence of the OPA1 gene mutant and the antisense primer can be used to amplify the PCR reaction , the sense and the antisense primer, Amplification can be used to diagnose optic atrophy or sensory motor neuropathy.

The term " probe " means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or a few hundred nucleotides that can specifically bind to mRNA, and is labeled to confirm the presence or absence of a specific mRNA . The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art. Therefore, hybridization using a probe complementary to the nucleotide sequence of the OPA1 gene mutant can be used to diagnose optic atrophy or sensory motor neuropathy through hybridization.

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

The term "antisense nucleic acid" refers to complementary with the sequence I molecules of the nucleic acid base to form a OPA1 gene mutant and dimer on OPA1 gene variants that are targeted, and may be used to detect the OPA1 gene variants.

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

Another aspect provides a kit for diagnosing the onset or the risk of developing optic atrophy or sensory motor neuropathy comprising the composition.

The kit comprises a protein consisting of an amino acid sequence comprising an L620fs or R905Q mutation in the amino acid sequence of SEQ ID NO: 1 or a protein consisting of a nucleotide sequence comprising 1857-1858 deLinsT or 2714G> A mutation in the nucleotide sequence of SEQ ID NO: 2, By detecting the nucleotide specifically, it is possible to diagnose the onset of optic nerve atrophy or sensory motor neuropathy or the risk of its onset.

Specifically, the kit may comprise an L620fs or R905Q mutation site of a protein consisting of an amino acid sequence comprising the L620fs or R905Q mutation in the amino acid sequence of SEQ ID NO: 1 or a mutation site of R905Q in the amino acid sequence of SEQ ID NO: 1, or 1857-1858delinsT or 2714G> A Specific detection of the 1857-1858 deLinsT or 2714G > A mutation site of a polynucleotide consisting of a nucleotide sequence including a mutation can diagnose the onset or the risk of the onset of optic nerve atrophy or sensory motor neuropathy.

The kit includes an antibody that specifically recognizes a protein consisting of an amino acid sequence comprising the L620fs or R905Q mutation in the amino acid sequence of SEQ ID NO: 1, or an antibody that specifically recognizes a nucleotide sequence of 1857-1858 deLinsT or 2714G> A mutation in the nucleotide sequence of SEQ ID NO: A probe or an antisense nucleic acid capable of specifically detecting a polynucleotide consisting of a sequence, and may further include one or more other component compositions, solutions or devices suitable for analysis.

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

The RT-PCR kit used RT-PCR to specifically detect the 1857-1858 deLinsT or 2714G> A mutation site of the polynucleotide consisting of the nucleotide sequence including 1857-1858 deLinsT or 2714G> A mutation in the nucleotide sequence of SEQ ID NO: Lt; RTI ID = 0.0 > a < / RTI > The RT-PCR kit may comprise a primer pair that is specific for the 1857-1858 deLinsT or 2714G> A mutation site of the polynucleotide consisting of the nucleotide sequence 1857-1858 delinsT or 2714G> A mutation in the nucleotide sequence of SEQ ID NO: 2, Other suitable containers, reaction buffers, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC-water, sterile water, and the like.

The microarray chip may comprise a DNA or RNA polynucleotide probe. The microarray is usually a polynucleotide comprising a probe specific for the 1857-1858 deLinsT or 2714G> A mutation site of a polynucleotide consisting of a nucleotide sequence comprising 1857-1858 deLinsT or 2714G > A mutation in the nucleotide sequence of SEQ ID NO: 2 Lt; RTI ID = 0.0 > microarray. ≪ / RTI > The microarray detects the presence of a 1857-1858 delins T or 2714 G> A mutation site of a polynucleotide consisting of a nucleotide sequence including 1857-1858 delins T or 2714G> A mutation in the nucleotide sequence of SEQ ID NO: 2 to detect optic atrophy or sensory motor nerve Can provide useful information to diagnose the likelihood of developing a pathology.

A method for preparing a microarray by immobilizing a probe for a 1857-1858 delins T or 2714 G> A mutation site of a polynucleotide consisting of a nucleotide sequence containing 1857-1858 delins T or 2714 G> A mutation in the nucleotide sequence of SEQ ID NO: 2 on a substrate Are well known in the art. For example, the DNA microarray can be obtained by a micropipetting method using a piezo electric method or a method using a spotter in the form of a pin, Alternatively, the probe for the 2714G > A mutation site can be immobilized on the substrate. The substrate of the microarray may be coated with an activating group selected from the group consisting of amino-silane, poly-L-lysine and aldehyde, but is not limited thereto . The substrate may be selected from the group consisting of slide glass, plastic, metal, silicon, nylon film, and nitrocellulose membrane, but is not limited thereto.

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

The protein chip kit can measure the expression level of a protein consisting of the amino acid sequence comprising the L620fs or R905Q mutation in the amino acid sequence of SEQ ID NO: The protein chip kit may comprise a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, or the like for immunological detection of the antibody. As the substrate, a nitrocellulose membrane, a 96-well plate synthesized with a polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, a slide glass made of glass, or the like can be used as the substrate, and a peroxidase ), Alkaline phosphatase, and the like can be used. As the fluorescent material, FITC, RITC and the like can be used. As the chromogenic substrate, ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid)), OPD (Tetramethylbenzidine), and the like can be used.

Another aspect is the use of an amino acid sequence comprising an L620fs or R905Q mutation in the amino acid sequence of SEQ ID NO: 1 in a biological sample isolated from an individual to diagnose optic atrophy or sensory motor neuropathy or to provide information for predicting the risk of onset Or a polynucleotide consisting of a nucleotide sequence comprising 1857-1858 deLinsT or 2714G> A mutation in the nucleotide sequence of SEQ ID NO: 2.

The term " individual " refers to an individual who seeks to determine the onset or onset of optic atrophy or sensory motor neuropathy or to predict the risk of onset. The subject may be a vertebrate, specifically a mammal, an amphibian, a reptile, a bird, or the like, and more specifically a mammal, for example, a human ( Homo sapiens ).

The term " biological sample " may include, but is not limited to, tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine samples separated from the subject.

The optic atrophy or sensory motor neuropathy may be caused by the c.1857-1858delinsT and c.2714G > A complex heterozygous mutations of the OPA1 gene. A c.1857-1858delinsT or c.2714G> A mutation in the OPA1 gene is a recessive mutation, but only one of them may not cause optic atrophy or sensory motor neuropathy.

Therefore, in the biological sample separated from the individual, the L620fs mutation site of the protein consisting of the amino acid sequence comprising the L620fs mutation in the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence comprising the R905Q mutation in the amino acid sequence of SEQ ID NO: In the presence of the R905Q mutation site, optic atrophy or sensory motor neuropathy may be or may be diagnosed as having a risk of onset.

Further, in the biological sample separated from the subject, 1857-1858 delins T mutation site of the polynucleotide consisting of the nucleotide sequence including the 1857-1858 delins T mutation in the nucleotide sequence of SEQ ID NO: 2 and 2714 G> A mutation in the nucleotide sequence of SEQ ID NO: 2 2714G > A mutation site of a polynucleotide consisting of a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 2 is present, optic atrophy or sensory motor neuropathy may be or may be diagnosed as being at risk.

The protein may be detected using an agent capable of specifically detecting the protein.

Specifically, the agent capable of specifically detecting the protein may be a preparation capable of specifically detecting L620fs or R905Q mutation sites.

The agent capable of specifically detecting the protein may be, but is not limited to, an antibody.

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

The polynucleotide may be detected using an agent capable of specifically detecting 1857-1858 delins T or 2714 G> A mutation site of the polynucleotide.

The agent capable of specifically detecting the 1857-1858 delins T or 2714 G> A mutation site of the polynucleotide may be, but is not limited to, a primer, a probe, or an antisense nucleic acid.

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

An OPA1 protein variant or OPA1 gene variant according to one aspect can be used as a marker for diagnosing optic atrophy or sensory motor neuropathy. In addition, it is possible to effectively predict or diagnose optic atrophy or sensory motor neuropathy using the marker, and further to develop a drug for treating optic atrophy or sensory motor neuropathy.

1 is a block diagram illustrating WES results performed in a sub-patient.
FIG. 2 is a diagram showing a chromatogram of OPA1 gene sequence analysis of a normal person and a patient. FIG.
Figure 3 is a human vertebrate species (Homo sapiens; NP_056375.2), mouse (Mus musculus; NP_598513.1), rats (Rattus norvegicus ; NP_598269.3), Bos Taurus (NP_001179890.1), Chicken ( Gallus gallus ; NP_001034398.1), Xenopus (Silurana) tropicalis ; NP_001120510.1), which is the result of conservation analysis of OPA1 mutation site and surrounding amino acid sequence.
Figure 4 shows the result of fundus examination of a family of II-1 patients: (A) shows generally pale optic discs in the right eye of II-1 patient, (B) shows pale optic discs in II- (C) shows that the father has a normal optic disc, and (D) shows that the mother has a normal optic disc.
FIG. 5 is a diagram showing a lower limb MRI result of patient II-1: (A) is a coronal image of lower limb MRI, and (B) to (D) is an axial image of lower limb MRI.
Figure 6 shows the histopathological test results of patient II-1: (A) is the result of toluidine blue staining optical microscope at x400 magnification, (B) shows the number of MFs according to MF diameter (Fiber number, a histogram showing the mm ^2), (C) is a x3,000 magnification electron microscope, (D) is an electron microscope magnification x10,000 result.

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

Example 1 . OPA1  Identification of complex heterozygous mutations in genes

1. Selection of patients

All three Korean households were tested on FC394. One of them was suffering from optic atrophy and sensorimotor neuropathy, and his parents showed normal phenotype. Parents were identified by genotyping 15 microsatellites using the PowerPlex 16 system (Promega Corporation, Madison, Wis., USA). In addition to this family, 300 healthy individuals were tested as controls. Written consent was obtained from all participants in accordance with the protocol approved by the Clinical Examination Committee of Sungkyunkwan University, Samsung Seoul Hospital (Seoul, Korea) and National Kongju University (Chungnam, Korea;

2. all Exome  Sequencing and identification of causative genes

Experiments were performed to identify the genes responsible for optic atrophy and sensory motor neuropathy in patients with optic atrophy and sensory motor neuropathy.

Specifically, three peripheral blood DNA fractions of FC394 family members were isolated using a QIAamp blood DNA purification kit (Qiagen GmbH, Hilden, Germany). Whole exome sequencing (WES) is an efficient molecular diagnostic tool and has recently been introduced into hereditary peripheral neuropathy. WES was performed using a SeqCap EZ human exome library version 3.0 (Roche NimbleGen, Inc., Madison, WI, USA) and a HiSeq 2000 genome analyzer (Illumina, Inc., San Diego, Calif. (Parent), I-2 (parent), and II-1 (patient) in the FC394 family according to the method described in J. Mutat. 33: 1610-1615, The UCSC assembly hg19 (NCBI build 37.1) was used as a reference sequence. In this study, mutants with more than 20 single nucleotide polymorphisms (SNPs) were used. Nonsense, nonsense, and exon insertion-deletion and splice site variants were selected from genetic peripheral neuropathy-associated genes and optic nerve atrophy-related genes, and the selected mutants were identified in the dbSNP142 database (http: www.ncbi.nlm.nih.gov), 1,000 genome project databases (http://www.1000genomes.org/) and the exomorph variant project (http://evs.gs.washington.edu/EVS/) .

Candidate mutants were confirmed by Sanger sequencing on I-1, I-2 and II-1 individuals using an ABI3130XL genetic analyzer (Applied Biosystem; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Mutations may occur when (i) they are inherited by a recessive pattern or are caused by a de novo mutation; (ii) when the allele is co-segregated with the diseased family members; (iii) not found in 300 healthy controls; And (iv) a cause mutation when not reported in the dbSNP142 database, the 1000 genome project database or the exomorph variant project. Genotype-phenotype correlations were also considered to determine candidate variants. The genomic evolutionary rate profiling (GERP) score was determined by the GERP ++ program (http://mendel.stanford.edu/SidowLab/down-loads/gerp/index.html). In silico analysis can be performed using SIFT (http://sift.jcvi.org), MUpro (http://www.ics.uci.edu/~baldig/mutation), and PolyPhen2 (http: // genetics. bwh.harvard.edu/pph2/) prediction algorithm.

1 is a block diagram illustrating WES results performed in a sub-patient. Table 1 shows the results of WES performed in sub-mother patients. FIG. 2 is a diagram showing a chromatogram of OPA1 gene sequence analysis of a normal person and a patient. FIG.

Figures 1 and 2, the patient is a new pair complex release bonding OPA1 mutations in other words, in the exon 20 at p.L620fs * 13 (c.1857-1858delinsT) and exon 27 mutation p.R905Q (c .2714G> A) mutation. These mutations were located in the intermediate domain and the guanosine triphosphatase (GTPase) effector domain, respectively. In the parental analysis, the mother was heterozygous for the p.R905Q missense mutation, and the father was heterozygous for the p.L620fs frame shift mutation. These two mutations were not detected in any of the 300 healthy Korean controls, the dbSNP138, 1000 genomic database, or the exosomal variant server.

I-1 (part) I-2 (mo) II-1 (patient) Overall Length (Gbp) 14,27 9,48 5.30 Mappable reads (%) 99.2 99.4 92.1 The range of the target area (? 1X,%) 95.7 95.2 97.9 The range of the target area (? 10X,%) 90.5 88.2 92.9 The average read depth (X) 58.7 40.6 57.9 Total number of SNPs 86,313 84,206 49,686 Number of coding SNPs 20,265 20,498 19,472 Insert - total number of deletions 7,902 7,472 6,898 Coding Insertion - Number of deletions 390 398 508

Figure 3 is a human vertebrate species (Homo sapiens; NP_056375.2), mouse (Mus musculus; NP_598513.1), rats (Rattus norvegicus ; NP_598269.3), Bos Taurus (NP_001179890.1), Chicken ( Gallus gallus ; NP_001034398.1), Xenopus (Silurana) tropicalis ; NP_001120510.1), which is the result of conservation analysis of OPA1 mutation site and surrounding amino acid sequence. As shown in Figure 3, the amino acid sequence around the mutation site was very well conserved among different vertebrate species.

Table 2 summarizes the OPA1 gene mutations and the psilocyko scores. Three silico analyzes, SIFT, PolyPhen2 and MUpro results, these mutations were predicted to be causative of the disease. In addition, the GERP score of the mutation was as high as 5.650 for c.1959C and 5.890 for c.2714G. Therefore, OPA1 This new complex heterozygosity mutation in the gene was considered to be responsible for the phenotype of the patient.

domain Change nucleotide Amino acid change In silico analysis GERP score SIFT PolyPhen2 MUpro MID c.1857-1858delinsT L620fs * 13 - - - 5.650 GED c.2714G> A R905Q 0.000 1,000 -0.630 5.890

Example  2. Optic Atrophy and Sensory motor neuropathy  Clinical Signs and Electrophysiological Evaluation of Patients

1. optic atrophy and Sensory motor neuropathy  Identification of patient's clinical signs

Experiments were performed to confirm clinical signs of optic atrophy and sensory motor neuropathy with p.L620fs * 13 (c.1857-1858delinsT) and p.R905Q (c.2714G> A) complex heterozygous mutations in OPA1.

Specifically, all subjects were thoroughly assessed by a team of skilled neurologists and ophthalmologists to measure the degree and severity of visual, auditory, and neurological disorders. Visual examination included funduscopic examination, color vision examination and automated field of view evaluation. Patients were tested for motion and sensory disturbance, deep tendon reflexes and muscle weakness by two independent neurologists and performed pure hearing test.

Table 3 shows the clinical characteristics of II-1 patients with optic atrophy and sensory motor neuropathy. This patient was born healthy with natural labor between Korean parents who are not relatives of each other. This patient visited the neurology department of Samsung Seoul Hospital (Seoul, Korea) for the gait disorder at the age of 10 years. At the age of one, he was diagnosed with congenital cataracts and underwent corrective surgery, at which time optic atrophy and nystagmus were found in his eyes. Optic atrophy continued until he was declared legally blind at the age of six. Until he was 2 years old, he could not walk without support, and he suffered severe ataxia and postural instability until he was 10 years old. Neurological examinations performed at 10 years of age showed predominantly muscle weakness and atrophy of both extremity muscles in the lower extremities. In addition, atrophic changes of bilateral pes cavus, steppage gait, and endogenous muscle and calf muscle were found. Sting sensation, tactile sensation, sense of position and sense of vibration were reduced. There were no reflexes of the knee and ankle, pyramidal signs and cerebellar dysfunction were not observed. At age 10, the patient could not count the number of fingers at a distance of up to 15 cm with one eye, but the light recognized.

Fig. 4 is a diagram showing the result of fundus examination of a family of II-1 patients. In FIG. 4, (A) shows pale optic discs generally in the right eye of a patient with II-1, and (B) shows an overall thin optic disc in the left eye of a patient with II-1. 4 (C) shows that the father has a normal optic disc, and (D) shows that the mother has a normal optic disc.

As shown in Fig. 4, extensive eye examination showed severe optic atrophy diffused in the eye of the II-1 patient.

As a result of the hearing test, the II-1 patient had normal hearing. It was also normal in laboratory tests such as glucose level, hepatic enzyme, and renal function, and ultrasonic cardiac examination showed normal copper dissection.

On the other hand, although the parents of this II-1 patient had OPA1 heterozygous mutations, no abnormalities were observed in ophthalmological and electrophysiological evaluations. In addition, there were no visual problems, and fundus examinations including optic nerve evaluation and official color test were normal.

race Korean French person French person French person French person American American German Reference Example 2 Bonneau et al. Schaaf et al. Pesch et al. gender male female female male male male female female Test Age
(years) 10 6 11 14 16 8 3 30 Age of onset 12 months 42 months 12 months 18 months 3 years old 12 months 6 months child Mutation Amino acid change R905Q
L620fs I382M
R557 ^a V402M
V903Gfs ^a I382M
R824 ^a I382M
E487K I382M
V903Gfs ^a I382M
V903Gfs ^a E270K
R290W domain GED
MID GTPase
MID GTPase
GED GTPase
MID GTPase
GTPase GTPase
GED GTPase
GED GTPase
GTPase Clinical signs Optic atrophy Overall Atrophy Yes Yes Yes Yes Serious spread Serious spread Overall Atrophy Vision Blind 0.01 / 0.01 Unknown 0.01 / 0.01 Blind Significantly reduced Unknown 0.2 / 0.2 Cataract Yes Unknown Unknown No Unknown No No No Peripheral neuropathy Yes Yes Yes Yes Yes Unknown Unknown Unknown Ataxia Yes Yes Yes Yes Yes Yes Yes Unknown test results VEP none continuing continuing continuing continuing Unknown Unknown Unknown NCS Axon sensory movement Axon sense Axon sensory movement Axon sense Axon sensory movement Unknown Unknown Unknown Brain MRI normal normal Cerebellar atrophy Cerebellar atrophy Cerebellar atrophy normal Unknown Unknown Not MRI Fat infiltration NA NA NA NA NA NA NA Neurobiology Axon nerve neuropathy NA NA NA NA NA NA NA

Bonneau et al., Brain 137: 3301, 2014;

Schaaf et al., Mol. Genet. Metab. 103: 383-387, 2011;

Pesch et al., Hum Mol Genet 10: 1359-1368, 2011;

a, translation termination codon;

GED, GTPase effector domain;

MID, intermediate domain;

VEP, visual evoked potentials;

NCS, nerve conduction studies;

MRI, magnetic resonance imaging.

2. Optic Atrophy and Sensory motor neuropathy  Electrophysiological evaluation of patients

Electrophysiological evaluation was performed on OPA1 patients with optic atrophy and sensory motor neuropathy with p.L620fs * 13 (c.1857-1858delinsT) and p.R905Q (c.2714G> A) complex heterozygous mutations.

Specifically, nerve conduction studies were performed on all limbs of the patient (Medtronic Keypoint 4; Medtronic Inc., Minneapolis, MN, USA). The motor nerve conduction velocities (MNCVs) of the median nerve and ulnar nerve were measured by stimulation at the elbow or wrist, while the composite of the abductor pollicis brevis and the adductor digiti quinti Compound muscle action potentials (CMAPs) were recorded. Similarly, MNCVs of the peroneal and tibial nerves were measured by knee and ankle stimulation, recording CMAPs for each of the extensor digitorum brevis and the adductor hallucis. The CMAP amplitude was measured from the baseline to the voice peak value. Sensory nerve conduction velocities were measured for the finger-wrist portion from the median nerve and ulnar nerve by orthodromic scoring and also recorded for the nervous nerves. Sensory nerve action potential (SNAP) amplitude was measured from the positive peak to the negative peak.

Table 4 shows the results of electrophysiological evaluations performed on II-1 patients with novel complex mutations in the OPA 1 gene. SNAPs were absent in all limbs when the II-1 patient was 6, 8, and 10 years of age. CMAPs decreased in the peroneal and tibial nerves, consistent with axonal polyneuropathy. When visual evoked potentials (VEP) were performed at 6 years of age, no dislocation was observed. However, brainstem auditory evoked potentials (BAEP) were normal. In parents, VEP and BAEP were normal.

Direction (6 years old) Direction (8 years old) Direction (10 years old) parameter Right side left Right side left Right side left Normal value Median nerve TL (ms) 3.0 2.8 3.4 2.9 3.3 3.0 <3.9 CMAP (mV) 7.9 8.8 7.3 6.9 6.2 7.6 > 6.0 MNCV (m / sec) 54.2 54.2 51.8 53.7 48.0 55.0 > 50.5 Ulnar nerve TL (ms) 2.4 2.4 2.4 2.8 2.8 2.6 <3.0 CMAP (mV) 8.0 6.1 7.4 6.7 8.1 7.4 > 8.0 MNCV (m / sec) 53.8 54.0 53.6 53.4 55.0 53.0 > 51.1 Peroneal nerve TL (ms) 2.3 2.9 2.7 3.0 3.8 3.6 <5.3 CMAP (mV) 1.1 1.3 1.1 1.0 0.6 1.7 > 1.6 MNCV (m / sec) 46.3 48.6 42.2 43.5 45.0 50.0 > 41.2 Tibial nerve TL (ms) 2.3 2.6 2.9 3.0 4.1 3.9 <5.4 CMAP (mV) 4.7 5.1 2.6 2.8 2.9 2.2 > 6.0 MNCV (m / sec) 45.5 46.3 42.0 43.8 41.0 45.0 > 41.1 Median sensory nerve SNAP (μV) A A A A A A > 8.8 SNCV (m / sec) A A A A A A > 39.3 Ulnar sensory nerve SNAP (μV) A A A A A A > 7.9 SNCV (m / sec) A A A A A A > 37.5 Sural nerve SNAP (μV) A A A A A A > 6.0 SNCV (m / sec) A A A A A A > 32.1

Bold, meaning an abnormal value;

A, no dislocation;

TL, terminal latency;

CMAP, compound muscle action potential;

MNCV, motor nerve conduction velocity;

SNAP, sensory nerve action potential;

SNCV, sensory nerve conduction velocity.

Example  3. Optic Atrophy and Sensory motor neuropathy  Magnetic resonance imaging scan of patient's lower limbs and brain

Magnetic resonance imaging (MRI) of the lower limb and brain in OPA1 patients with optic atrophy with p.L620fs * 13 (c.1857-1858delinsT) and p.R905Q (c.2714g> A) complex heterozygous mutations Photographing was performed.

Specifically, patient II-1 was measured at follow-up visits at age 8 using a 3.0-T system (Siemens AG, Munich, Germany). The lower limb is the axial face (field of view: FOV, 24-32 cm; Section thickness, 6 mm; Section interval, 0.5-1.0 mm] and coronal plane [FOV, 38-40 cm; Section thickness, 4-5 mm; Section interval, 0.5-1.0 mm]. T1-weighted spin-echo (SE) [repetition time (TR) / echo time (TE) 570-650 / 14-20, 512 matrix] TE 2800-4000 / 96-99, 512 matrix) and fat-suppressed T2-weighted SE (TR / TE 3090-4900 / 85-99, 512 matrix) protocol.

5 is a diagram showing the results of the lower limb MRI of the patient II-1. 5 (A) is a coronal image of the lower limb MRI, and (B) to (D) is an axial image of the lower limb MRI.

As shown in Fig. 5, T1-weighted MRIs taken at 10 years of age showed muscle atrophy and fat substitution in the lower calf muscles, but the hips and thigh muscles were near normal. These results were consistent with the theory of length - dependent axon degeneration. In addition, fat replacement of the calf muscle prevailed in the anterior portion, including the anterior tibial muscle, consistent with the foot drop and steppage gait of this patient. On the other hand, from brain MRI performed at the age of 8 years, the cerebral cortex and cerebellum were normal in structure and size.

Example  4. Histopathological analysis of patients with optic atrophy

Biopsy was performed to identify histopathologic symptoms in OPA1 patients with optic atrophy with p.L620fs * 13 (c.1857-1858delinsT) and p.R905Q (c.2714G> A) complex heterozygous mutations Respectively.

Specifically, histopathologic analysis of distal contralateral nerve biopsy was performed at age 6 for patient II-1. 2% glutaraldehyde (pH 7.4; Sigma-Aldrich, St. Louis, Mo.) dissolved in 0.025 M cacodylate buffer was used in addition to the optical microscopy test (BX51; Olympus Corporation, MA, USA), and processed for semi-thin and ultra-thin studies. &Lt; tb &gt; &lt; TABLE &gt; The semi-thin portion was stained with toluidine blue (Sigma-Aldrich) for evaluation under an optical microscope. The ultra-thin portion (60-65 nm) was stained with uranium acetate and lead citrate (Sigma-Aldrich) for a microstructure study (H-7650; Hitachi, Ltd., Tokyo, Japan). The g-ratio of myelinated fibers (MFs), axon diameter, myelin thickness and MFs was measured from a semi-thin cross section using a computer-based image analyzer (AnalySIS, Olympus Soft Imaging Solutions, Respectively.

Figure 6 is a histopathological test result of Patient II-1. 6A is a histogram showing the number of MFs (fiber number / mm ² ) according to MF diameter (fiber diameter, μm), and FIG. 6C is a histogram x3,000 magnification electron microscope results, and (D) x10,000 magnification electron microscopic results.

As shown in FIG. 6A, the semi-thin cross section of the distal appendicular nerve showed moderate and small MFs with complete loss of large MFs. In a normal 2 year old woman, a healthy distal distal nerve MF number was measured as 4,245 MFs / mm ² , compared with 14,170 ± 2,250 / mm ² . The range and mean of the MF diameters were 2.5-10.3 μm and 5.0 μm, respectively. As shown in FIG. 6B, the histogram showed a unimodal distribution pattern, with MFs having a diameter of 8 μm or more constituting 0.9% of the total population. In this case, the MF% area was 8.8%. Remaining MFs showed occasional excessive folding of the few seconds and little evidence of regeneration. No evidence of dehydration, rehydration or onion bulb formation was seen.

As shown in FIG. 6C, as a result of the electron microscopic examination, the remaining MFs had focal abnormalities of a few seconds, including irregular aquatic meets, malformation type, or focal noncompaction. As shown in Fig. 6D, the anterior axon was relatively well preserved in a relatively sparse state. In addition, fibroblast proliferation of the nerve endocardium and large amounts of collagen deposition were recorded.

<110> Samsung Life Public Welfare Foundation          KONGJU NATIONAL UNIVERSITY INDUSTRY-UNIVERSITY COOPERATION FOUNDATION <120> OPA1 as a causative gene of optic atrophy or sensorimotor          neuropathy and method for diagnosing the disease using the same <130> PN115307KR <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 960 <212> PRT <213> Homo sapiens <400> 1 Met Trp Arg Leu Arg Arg Ala Ala Val Ala Cys Glu Val Cys Gln Ser   1 5 10 15 Leu Val Lys His Ser Ser Gly Ile Lys Gly Ser Leu Pro Leu Gln Lys              20 25 30 Leu His Leu Val Ser Ser Ser Ile Tyr His Ser His His Pro Thr Leu          35 40 45 Lys Leu Gln Arg Pro Gln Leu Arg Thr Ser Phe Gln Gln Phe Ser Ser      50 55 60 Leu Thr Asn Leu Pro Leu Arg Lys Leu Lys Phe Ser Pro Ile Lys Tyr  65 70 75 80 Gly Tyr Gln Pro Arg Arg Asn Phe Trp Pro Ala Arg Leu Ala Thr Arg                  85 90 95 Leu Leu Lys Leu Arg Tyr Leu Ile Leu Gly Ser Ala Val Gly Gly Gly             100 105 110 Tyr Thr Ala Lys Lys Thr Phe Asp Gln Trp Lys Asp Met Ile Pro Asp         115 120 125 Leu Ser Glu Tyr Lys Trp Ile Val Pro Asp Ile Val Trp Glu Ile Asp     130 135 140 Glu Tyr Ile Asp Phe Glu Lys Ile Arg Lys Ala Leu Pro Ser Ser Glu 145 150 155 160 Asp Leu Val Lys Leu Ala Pro Asp Phe Asp Lys Ile Val Glu Ser Leu                 165 170 175 Ser Leu Leu Lys Asp Phe Phe Thr Ser Gly Ser Pro Glu Glu Thr Ala             180 185 190 Phe Arg Ala Thr Asp Arg Gly Ser Glu Ser Asp Lys His Phe Arg Lys         195 200 205 Val Ser Asp Lys Glu Lys Ile Asp Gln Leu Gln Glu Glu Leu Leu His     210 215 220 Thr Gln Leu Lys Tyr Gln Arg Ile Leu Glu Arg Leu Glu Lys Glu Asn 225 230 235 240 Lys Glu Leu Arg Lys Leu Val Leu Gln Lys Asp Asp Lys Gly Ile His                 245 250 255 His Arg Lys Leu Lys Lys Ser Leu Ile Asp Met Tyr Ser Glu Val Leu             260 265 270 Asp Val Leu Ser Asp Tyr Asp Ala Ser Tyr Asn Thr Gln Asp His Leu         275 280 285 Pro Arg Val Val Val Gly Asp Gln Ser Ala Gly Lys Thr Ser Val     290 295 300 Leu Glu Met Ile Ala Gln Ala Arg Ile Phe Pro Arg Gly Ser Gly Glu 305 310 315 320 Met Met Thr Arg Ser Pro Val Lys Val Thr Leu Ser Glu Gly Pro His                 325 330 335 His Val Ala Leu Phe Lys Asp Ser Ser Arg Glu Phe Asp Leu Thr Lys             340 345 350 Glu Glu Asp Leu Ala Ala Leu Arg His Glu Ile Glu Leu Arg Met Arg         355 360 365 Lys Asn Val Lys Glu Gly Cys Thr Val Ser Pro Glu Thr Ile Ser Leu     370 375 380 Asn Val Lys Gly Pro Gly Leu Gln Arg Met Val Leu Val Asp Leu Pro 385 390 395 400 Gly Val Ile Asn Thr Val Thr Ser Gly Met Ala Pro Asp Thr Lys Glu                 405 410 415 Thr Ile Phe Ser Ile Ser Lys Ala Tyr Met Gln Asn Pro Asn Ala Ile             420 425 430 Ile Leu Cys Ile Gln Asp Gly Ser Val Asp Ala Glu Arg Ser Ile Val         435 440 445 Thr Asp Leu Val Ser Gln Met Asp Pro His Gly Arg Arg Thr Ile Phe     450 455 460 Val Leu Thr Lys Val Asp Leu Ala Glu Lys Asn Val Ala Ser Ser Ser 465 470 475 480 Arg Ile Gln Gln Ile Ile Glu Gly Lys Leu Phe Pro Met Lys Ala Leu                 485 490 495 Gly Tyr Phe Ala Val Val Thr Gly Lys Gly Asn Ser Ser Glu Ser Ile             500 505 510 Glu Ala Ile Arg Glu Tyr Glu Glu Glu Phe Phe Gln Asn Ser Lys Leu         515 520 525 Leu Lys Thr Ser Met Leu Lys Ala His Gln Val Thr Thr Arg Asn Leu     530 535 540 Ser Leu Ala Val Ser Asp Cys Phe Trp Lys Met Val Val Arg Glu Ser Val 545 550 555 560 Glu Gln Gln Ala Asp Ser Phe Lys Ala Thr Arg Phe Asn Leu Glu Thr                 565 570 575 Glu Trp Lys Asn Asn Tyr Pro Arg Leu Arg Glu Leu Asp Arg Asn Glu             580 585 590 Leu Phe Glu Lys Ala Lys Asn Glu Ile Leu Asp Glu Val Ile Ser Leu         595 600 605 Ser Gln Val Thr Pro Lys His Trp Glu Glu Ile Leu Gln Gln Ser Leu     610 615 620 Trp Glu Arg Val Ser Thr His Val Ile Glu Asn Ile Tyr Leu Pro Ala 625 630 635 640 Ala Gln Thr Met Asn Ser Gly Thr Phe Asn Thr Thr Val Asp Ile Lys                 645 650 655 Leu Lys Gln Trp Thr Asp Lys Gln Leu Pro Asn Lys Ala Val Glu Val             660 665 670 Ala Trp Glu Thr Leu Gln Glu Glu Phe Ser Arg Phe Met Thr Glu Pro         675 680 685 Lys Gly Lys Glu His Asp Asp Ile Phe Asp Lys Leu Lys Glu Ala Val     690 695 700 Lys Glu Glu Ser Ile Lys Arg His Lys Trp Asn Asp Phe Ala Glu Asp 705 710 715 720 Ser Leu Arg Val Ile Gln His Asn Ala Leu Glu Asp Arg Ser Ser Ser                 725 730 735 Asp Lys Gln Gln Trp Asp Ala Ile Tyr Phe Met Glu Glu Ala Leu             740 745 750 Gln Ala Arg Leu Lys Asp Thr Glu Asn Ala Ile Glu Asn Met Val Gly         755 760 765 Pro Asp Trp Lys Lys Arg Trp Leu Tyr Trp Lys Asn Arg Thr Gln Glu     770 775 780 Gln Cys Val His Asn Glu Thr Lys Asn Glu Leu Glu Lys Met Leu Lys 785 790 795 800 Cys Asn Glu Glu His Pro Ala Tyr Leu Ala Ser Asp Glu Ile Thr Thr                 805 810 815 Val Arg Lys Asn Leu Glu Ser Arg Gly Val Glu Val Asp Pro Ser Leu             820 825 830 Ile Lys Asp Thr Trp His Gln Val Tyr Arg Arg His Phe Leu Lys Thr         835 840 845 Ala Leu Asn His Cys Asn Leu Cys Arg Arg Gly Phe Tyr Tyr Tyr Gln     850 855 860 Arg His Phe Val Asp Ser Glu Leu Glu Cys Asn Asp Val Val Leu Phe 865 870 875 880 Trp Arg Ile Gln Arg Met Leu Ala Ile Thr Ala Asn Thr Leu Arg Gln                 885 890 895 Gln Leu Thr Asn Thr Glu Val Arg Arg Leu Glu Lys Asn Val Lys Glu             900 905 910 Val Leu Glu Asp Phe Ala Glu Asp Gly Glu Lys Lys Ile Lys Leu Leu         915 920 925 Thr Gly Lys Arg Val Gln Leu Ala Glu Asp Leu Lys Lys Val Arg Glu     930 935 940 Ile Gln Glu Lys Leu Asp Ala Phe Ile Glu Ala Leu His Gln Glu Lys 945 950 955 960 <210> 2 <211> 2883 <212> DNA <213> Homo sapiens <400> 2 atgtggcgac tacgtcgggc cgctgtggcc tgtgaggtct gccagtcttt agtgaaacac 60 agctctggaa taaaaggaag tttaccacta caaaaactac atctggtttc acgaagcatt 120 tatcattcac atcatcctac cttaaagctt caacgacccc aattaaggac atcctttcag 180 cagttctctt ctctgacaaa ccttccttta cgtaaactga aattctctcc aattaaatat 240 ggctaccagc ctcgcaggaa tttttggcca gcaagattag ctacgagact cttaaaactt 300 cgctatctca tactaggatc ggctgttggg ggtggctaca cagccaaaaa gacttttgat 360 cagtggaaag atatgatacc ggaccttagt gaatataaat ggattgtgcc tgacattgtg 420 tgggaaattg atgagtatat cgattttgag aaaattagaa aagcccttcc tagttcagaa 480 gaccttgtaa agttagcacc agactttgac aagattgttg aaagccttag cttattgaag 540 gactttttta cctcaggttc tccggaagaa acggcgttta gagcaacaga tcgtggatct 600 gaaagtgaca agcattttag aaaggtgtca gacaaagaga aaattgacca acttcaggaa 660 gaacttctgc acactcagtt gaagtatcag agaatcttgg aacgattaga aaaggagaac 720 aaagaattga gaaaattagt attgcagaaa gatgacaaag gcattcatca tagaaagctt 780 aagaaatctt tgattgacat gtattctgaa gttcttgatg ttctctctga ttatgatgcc 840 agttataata cgcaagatca tctgccacgg gttgttgtgg ttggagatca gagtgctgga 900 aagactagtg tgttggaaat gattgcccaa gctcgaatat tcccaagagg atctggggag 960 atgatgacac gttctccagt taaggtgact ctgagtgaag gtcctcacca tgtggcccta 1020 tttaaagata gttctcggga gtttgatctt accaaagaag aagatcttgc agcattaaga 1080 catgaaatag aacttcgaat gaggaaaaat gtgaaagaag gctgtaccgt tagccctgag 1140 accatatcct taaatgtaaa aggccctgga ctacagagga tggtgcttgt tgacttacca 1200 ggtgtgatta atactgtgac atcaggcatg gctcctgaca caaaggaaac tattttcagt 1260 atcagcaaag cttacatgca gaatcctaat gccatcatac tgtgtattca agatggatct 1320 gtggatgctg aacgcagtat tgttacagac ttggtcagtc aaatggaccc tcatggaagg 1380 agaaccatat tcgttttgac caaagtagac ctggcagaga aaaatgtagc cagtccaagc 1440 aggattcagc agataattga aggaaagctc ttcccaatga aagctttagg ttattttgct 1500 gttgtaacag gaaaagggaa cagctctgaa agcattgaag ctataagaga atatgaagaa 1560 gagttttttc agaattcaaa gctcctaaag acaagcatgc taaaggcaca ccaagtgact 1620 acaagaaatt taagccttgc agtatcagac tgcttttgga aaatggtacg agagtctgtt 1680 gaacaacagg ctgatagttt caaagcaaca cgttttaacc ttgaaactga atggaagaat 1740 aactatcctc gcctgcggga acttgaccgg aatgaactat ttgaaaaagc taaaaatgaa 1800 atccttgatg aagttatcag tctgagccag gttacaccaa aacattggga ggaaatcctt 1860 caacaatctt tgtgggaaag agtatcaact catgtgattg aaaacatcta ccttccagct 1920 gcgcagacca tgaattcagg aacttttaac accacagtgg atatcaagct taaacagtgg 1980 actgataaac aacttcctaa taaagcagta gaggttgctt gggagaccct acaagaagaa 2040 ttttcccgct ttatgacaga accgaaaggg aaagagcatg atgacatatt tgataaactt 2100 aaagaggctg ttaaggaaga aagtattaaa cgacacaagt ggaatgactt tgcggaggac 2160 agcttgaggg ttattcaaca caatgctttg gaagaccgat ccatatctga taaacagcaa 2220 tgggatgcag ctatttattt tatggaagag gctctgcagg ctcgtctcaa ggatactgaa 2280 aatgcaattg aaaacatggt gggtccagac tggaaaaaga ggtggttata ctggaagaat 2340 cggacccaag aacagtgtgt tcacaatgaa accaagaatg aattggagaa gatgttgaaa 2400 tgtaatgagg agcacccagc ttatcttgca agtgatgaaa taaccacagt ccggaagaac 2460 cttgaatccc gaggagtaga agtagatcca agcttgatta aggatacttg gcatcaagtt 2520 tatagaagac attttttaaa aacagctcta aaccattgta acctttgtcg aagaggtttt 2580 tattactacc aaaggcattt tgtagattct gagttggaat gcaatgatgt ggtcttgttt 2640 tggcgtatac agcgcatgct tgctatcacc gcaaatactt taaggcaaca acttacaaat 2700 actgaagtta ggcgattaga gaaaaatgtt aaagaggtat tggaagattt tgctgaagat 2760 ggtgagaaga agattaaatt gcttactggt aaacgcgttc aactggcgga agacctcaag 2820 aaagttagag aaattcaaga aaaacttgat gctttcattg aagctcttca tcaggagaaa 2880 taa 2883

Claims (20)

In the amino acid sequence of SEQ ID NO: 1,
OPA1 (optic atrophy 1) protein variant consisting of an amino acid sequence containing a mutation (L620fs) in which framehift occurs with leucine (L) at position 620. The variant of claim 1, wherein the frame shift is an OPA1 protein variant (SEQ ID NO: 2) wherein two cytosines (C) at positions 1857 and 1858 are deleted in the nucleotide sequence of SEQ ID NO: 2 and one thymine . A polynucleotide encoding an OPA1 protein variant according to claim 1. 4. The method of claim 3, wherein in the nucleotide sequence of SEQ ID NO: 2,
A polynucleotide consisting of a nucleotide sequence comprising a mutation (1857-1858 delins T) in which two cytosines (C) at positions 1857 and 1858 are deleted and one thymine (T) is inserted at that position. A preparation capable of specifically detecting a protein consisting of the amino acid sequence comprising the L620fs mutation in the amino acid sequence of SEQ ID NO: 1 and a protein consisting of the amino acid sequence comprising the R905Q mutation in the amino acid sequence of SEQ ID NO: 1 are specifically detected A formulation capable of; or
A preparation capable of specifically detecting a polynucleotide consisting of a nucleotide sequence comprising 1857-1858 delins T mutation in the nucleotide sequence of SEQ ID NO: 2 and a polynucleotide consisting of a nucleotide sequence comprising 2714G> A mutation in the nucleotide sequence of SEQ ID NO: 2 RTI ID = 0.0 &gt; a &lt; / RTI &gt;
A composition for diagnosing the onset or the risk of the onset of optic atrophy or sensory motor neuropathy. The composition according to claim 5, wherein the agent capable of specifically detecting the protein is an antibody. The composition according to claim 5, wherein the agent capable of specifically detecting the polynucleotide is an agent capable of specifically detecting 1857-1858 deLinsT or 2714G> A mutation site. The composition according to claim 5, wherein the agent capable of specifically detecting the polynucleotide is a primer, a probe, or an antisense nucleic acid. 9. The composition of claim 8, wherein the antisense nucleic acid is complementary to the polynucleotide or fragment thereof. 10. The composition of claim 9, wherein the fragment has at least 10 nucleotides. A kit for diagnosing the onset or risk of developing optic atrophy and sensory motor neuropathy comprising the composition of any one of claims 5 to 10. 12. The kit of claim 11, wherein the kit is an RT-PCR kit, a microarray chip kit, or a protein chip kit. In order to provide information for diagnosing optic atrophy or sensory motor neuropathy or for predicting the risk of onset, in a biological sample separated from an individual,
A protein consisting of an amino acid sequence comprising the L620fs or R905Q mutation in the amino acid sequence of SEQ ID NO: 1, or
1. A method for detecting a polynucleotide consisting of a nucleotide sequence comprising 1857-1858 deLinsT or 2714G > A mutation in the nucleotide sequence of SEQ ID NO: 2,
Wherein said optic atrophy or sensory motor neuropathy is caused by c.1857-1858delinsT and c.2714G > A complex heterozygous mutations of the OPA1 gene. delete 14. The method of claim 13, wherein the protein is detected using an agent capable of specifically detecting the protein. 16. The method of claim 15, wherein the agent capable of specifically detecting the protein is an antibody. The method according to claim 13, wherein the assay for detecting the protein is selected from the group consisting of Western blotting, ELISA, radioimmunoassay, radioimmunoprecipitation, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, Complement fixation assay, FACS or protein chip. 14. The method of claim 13, wherein the polynucleotide is detected using an agent capable of specifically detecting the 1857-1858 delins T or 2714 G> A mutation site of the polynucleotide. 19. The method according to claim 18, wherein the agent capable of specifically detecting the 1857-1858 delins T or 2714 G> A mutation site of the polynucleotide is a primer, a probe or an antisense nucleic acid. 14. The method according to claim 13, wherein the assay for detecting the polynucleotide is a reverse transcription polymerase chain reaction, a competitive reverse transcription polymerase chain reaction, a real time reverse transcription polymerase chain reaction, an RNase protection assay (RPA), a Northern blotting or a DNA chip.

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