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WO2019078684A2 - Marqueur de tmem43 destiné au diagnostic de la perte d'audition neurosensorielle et son utilisation - Google Patents

Marqueur de tmem43 destiné au diagnostic de la perte d'audition neurosensorielle et son utilisation Download PDF

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Publication number
WO2019078684A2
WO2019078684A2 PCT/KR2018/012442 KR2018012442W WO2019078684A2 WO 2019078684 A2 WO2019078684 A2 WO 2019078684A2 KR 2018012442 W KR2018012442 W KR 2018012442W WO 2019078684 A2 WO2019078684 A2 WO 2019078684A2
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nucleotide sequence
mutation
tmem43
genomic dna
polynucleotide
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Korean (ko)
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WO2019078684A9 (fr
WO2019078684A3 (fr
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최병윤
오두이
이창준
장민우
오수진
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Korea Institute of Science and Technology KIST
Seoul National University Hospital
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Korea Institute of Science and Technology KIST
Seoul National University Hospital
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Publication of WO2019078684A3 publication Critical patent/WO2019078684A3/fr
Publication of WO2019078684A9 publication Critical patent/WO2019078684A9/fr
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • compositions for the diagnosis of sensory neural deafness disorders for detecting genetic mutations for detecting genetic mutations, methods for detecting mutations in the genome to provide information about the diagnosis of sensory neural deafness, methods for predicting the prognosis for wort transplantation in patients with sensory nerve impairment
  • the present invention relates to a method for detecting a mutation of a genome to provide information for predicting a prognosis for a woW transplantation of a sensory neuron deaf hearing patient, and a sensory neuronal hearing loss disease model.
  • Congenital hearing loss occurs in about 3 out of 1,000 neonates, and more than 50% is known to be caused by genetic factors. Hereditary hearing loss is congenital and occurs from birth. There are many types of hearing loss, but the most common is sensorineural hearing loss (SNHL).
  • SNHL sensorineural hearing loss
  • Sensory nerve impairment refers to hearing loss caused by an abnormality in the function of detecting the sound of the cochlea or of an auditory nerve or a central nervous system that transmits a stimulation by sound to the brain. Sensory nerve impairment is classified into syndrome and non-syndrome according to the symptoms. Syndrome Sensory nerve impairment refers to the clinical symptoms or symptoms of other organs other than those caused by abnormal function of inner ear. It accounts for 30% of hereditary hearing loss, and over 300 types of syndromic hearing loss have been reported to date. Because it is easily classified as a characteristic symptom or anomaly, the causative gene can be easily traced in patients with the same cause.
  • Non-syndrome Sensory-nerve impairment refers to cases in which there are no abnormal symptoms or symptoms of other organs other than internal dysfunction, and only hearing impairment. It accounts for 70% of hereditary deafness, has a variety of causative genes, and strategic genetic diagnosis is required based on clinical information such as type of hearing loss, genetic type, and so on. Non-syndromic hearing loss is known to occur in 80% of genetic forms, 17% of dominant inheritance, 2 to 3% of X-linked, and 1% of mitochondrial hereditary genetic patterns.
  • Sensory nerve impairment is a common sensory neural deafness problem in the outer ear cells of the wah and auditory neuropathy in the inner ear cells of the wah or in the nerve itself.
  • auditory neuropathy exhibits an unidirectional or cochlear microphonic (CM) action, but no auditory brainstem response or very abnormal findings. Auditory neuropathy occurs sporadically in many patients, but may also be genetic. In autosomal dominant inheritance pattern, progressive hearing loss is present and accompanied by peripheral neuropathy. The autosomal recessive inheritance pattern generally presents with severe hearing loss in infancy and does not accompany peripheral neuropathy.
  • CM cochlear microphonic
  • compositions for the diagnosis of sensory neural deafness comprising a polynucleotide comprising a contiguous nucleotide sequence selected from the nucleotide sequence of the exon region of the TMEM43 gene or a complementary polynucleotide thereof for detecting a TMEM43 mutation.
  • Another aspect includes identifying a nucleotide sequence of a genomic DNA isolated from an individual; And comparing the confirmed nucleotide sequence of the genomic DNA with a standard nucleotide sequence to identify the mutation of the genomic DNA.
  • Another aspect is the use of a polynucleotide comprising a contiguous nucleotide sequence selected from the nucleotide sequence of the exon region of the TMEM43 gene to detect a TMEM43 mutation, or a polynucleotide comprising its complementary polynucleotide, ≪ / RTI >
  • Another aspect includes identifying a nucleotide sequence of a genomic DNA isolated from an individual; And comparing the identified nucleotide sequence of the genomic DNA with a standard nucleotide sequence to identify the mutation of the genomic DNA.
  • identifying a nucleotide sequence of a genomic DNA isolated from an individual and comparing the identified nucleotide sequence of the genomic DNA with a standard nucleotide sequence to identify the mutation of the genomic DNA.
  • Another aspect provides a sensory neural deafness disorder model comprising TMEM43 mutations.
  • compositions for the diagnosis of sensory neural deafness comprising a polynucleotide comprising a contiguous nucleotide sequence selected from the nucleotide sequence of the exon region of the TMEM43 gene or a complementary polynucleotide thereof for detecting a TMEM43 mutation.
  • Transmembrane protein 43 is a protein that functions to maintain the nuclear membrane structure by forming a protein complex in the inner nuclear membrane.
  • the TMEM43 may be a protein encoded by the TMEM43 gene.
  • GenBank Accession No. A polypeptide comprising the amino acid sequence of NP_077310.1 or a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • the TMEM43 gene includes a polynucleotide including a nucleotide sequence encoding the amino acid sequence of TMEM43, a polynucleotide including a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, A polynucleotide comprising a nucleotide sequence of NM_024334, a nucleotide sequence of NM_024334.2, or a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 2.
  • the position and sequence of the mutation can be easily confirmed using the registration number.
  • the specific sequence corresponding to the number registered in the UCSC genome browser or GenBank may change over time. It will be apparent to those of ordinary skill in the art that the scope of the present invention also affects the altered sequence.
  • the TMEM 43 may be expressed in the inner ear.
  • the TMEM 43 may be expressed in inner ear hair cells or supporting cells around the inner hair cells.
  • the TMEM 43 may be one that promotes spontaneous activity in the developing inner ear and induces survival and maturation of the auditory source cells prior to the onset of auditory evoked potentials.
  • the sensory neural deafness refers to hearing loss caused by an abnormality in the function of sensing the sound of the cochlea or of an auditory nerve or a central nervous system which transmits a stimulation by sound to the brain.
  • the sensory neural deafness may be non-syndromic sensorineural hearing loss (NS-SNHL).
  • Non-syndromic sensory neural deafness refers to hearing loss that does not exhibit abnormal symptoms or symptoms of other organs other than abnormal inner ear function.
  • the sensory neural deafness may be an auditory neuropathy.
  • the auditory neuropathy is a hearing disorder caused by a disorder in the synchrony of action potentials occurring in the auditory nerve fibers in the auditory stimulus.
  • the auditory neuropathy is an auditory neuropathy, Pathologically, the function of the outer hair cell is preserved and is due to the abnormalities of the inner hair cell and the auditory transmission pathway through the type 1 auditory nerve cell.
  • polynucleotide refers to a nucleotide polymer of any length, which polynucleotide can be used interchangeably with nucleic acids, or oligonucleotides.
  • the polynucleotide may specifically bind to the nucleotide sequence of the target region. Using the specific binding characteristics of such polynucleotides, it is possible to effectively isolate a target gene or a fragment thereof containing a target region from a mixture.
  • the polynucleotides may be singular or plural and may be in the form of a single strand or a double strand.
  • the polynucleotide may also be modified so that the sugar, base or phosphate site of a natural nucleotide, an analogue of a natural nucleotide, or a natural nucleotide, as well as being composed of a natural nucleotide, may be modified so long as it has a property of being hybridizable with a complementary nucleotide by hydrogen bonding (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)), and nucleotides selected from the group consisting of .
  • the polynucleotide may be DNA or RNA.
  • contiguous nucleotide sequence refers to two or more contiguous nucleotide sequences.
  • complementary means having complementarity enough to selectively hybridize to the nucleotide sequence under any particular hybridization or annealing conditions.
  • the polynucleotide may be a primer or a probe.
  • the primer or probe may be a nucleotide sequence perfectly complementary to the nucleotide sequence, but a nucleotide sequence that is substantially complementary may be used so long as it does not specifically interfere with hybridization .
  • the primer or the probe may have a modified nucleotide sequence within a range that does not interfere specifically with hybridization with respect to the nucleotide sequence of the target region.
  • primer means a single strand of oligonucleotides that can act as a starting point in the polymerization of a nucleotide by a polymerase.
  • the appropriate length of the primer may vary depending on various factors, for example, temperature and use of the primer.
  • the primer may have a length of 5 to 100 nt, 5 to 70 nt, 10 to 50 nt, or 15 to 30 nt.
  • the shorter the length of the primer the more stable hybridization complex can be formed with the template at a lower annealing temperature.
  • the primer is economical and has a size optimized for gene mutation detection.
  • the design of the primer can be easily carried out by a conventional technician with reference to the nucleotide sequence of the target region to be amplified. For example, it can be designed using a commercially available program for primer design. Examples of commercially available programs for primer design include the PRIMER 3 program.
  • the primer may further comprise a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, a peptide nucleic acid or an intercalating agent. Further, it may further comprise a labeling substance that emits fluorescence, phosphorescence, or radioactivity.
  • the fluorescent labeling substance may be VIC, NED, FAM, PET, or a combination thereof.
  • the labeling substance may be labeled at the 5 ' end of the polynucleotide.
  • the radioactive labeling substance may be incorporated into the amplification product through a PCR reaction using a polymerase chain reaction (PCR) in which a radioactive isotope such as 32 P or 35 S is added.
  • PCR polymerase chain reaction
  • the primers can be used for allele-specific PCR, PCR extension analysis, PCR-single strand conformation polymorphism (PCR-SSCP), TaqMan method and sequencing. have.
  • probe means a polynucleotide that is capable of sequence-specifically binding to a complementary polynucleotide strand.
  • the probe may have a length of 5 to 100 nt, 10 to 90 nt, 15 to 80 nt, 20 to 70 nt, or 30 to 50 nt. If the length of the probe is less than 10 nt, the accuracy for capturing the target area is low, and if the length is more than 100 nt, the synthesis cost is increased. Therefore, the probe is economical and has a size optimized for gene mutation detection.
  • the probe may be, for example, selected from the group consisting of a perfect match probe consisting of a sequence complementary to the nucleotide sequence of the target region and a probe having a sequence completely complementary to all nucleotide sequences except for the mutation position .
  • the probe may be used in hybridization methods such as microarray, Southern blotting, dynamic allele-specific hybridization, and DNA chip.
  • the microarray is used in a manner known in the art, for example, a group of probes or probes immobilized on a plurality of divided regions on a substrate.
  • the substrate can be any suitable rigid or semi-rigid support, such as a membrane, filter, chip, slide, wafer, fiber, magnetic bead or non-magnetic bead, gel, tubing, Microparticles, and capillaries.
  • the probe or a complementary probe thereof may be used in a method capable of hybridizing with a nucleic acid obtained from an individual and measuring the hybridization degree obtained therefrom.
  • gene refers to a structural unit that determines genetic information, and includes a structural gene having information for determining the base sequence of the protein's amino acid sequence or functional RNA (tRNA, rRNA, etc.), and / For example, a promoter, a repressor, an operator, and the like.
  • gene is understood to mean a single stranded side comprising a nucleotide sequence that is transcribed to produce a product of the gene.
  • the " nucleotide sequence of a gene &quot may be a nucleotide sequence that controls the expression of a nucleotide sequence and / or a structural gene contained in a single strand containing a nucleotide sequence to be transcribed to produce a product of the gene.
  • exon refers to a region containing information on the synthesis of a protein in the DNA sequence, for example, a nucleic acid molecule encoding part or all of the expressed protein.
  • the mutation may be one having a variation of the nucleotide sequence relative to the standard genomic DNA.
  • the mutation of the nucleotide sequence may include one or more nucleotide sequence substitution, insertion, duplication, and deletion (insertion and deletion: also referred to as 'InDel') with respect to the standard genomic DNA, , Translocation, and the like.
  • the substitution of the one or more nucleotide sequences may be, for example, a single nucleotide variant (SNV).
  • the mutation may be an autosomal dominant mutation.
  • the mutation may be a mutation of the exon region of the TMEM43 gene, for example, the 12th exon region.
  • the mutation may be the c.C1114T mutation in the nucleotide sequence of the TMEM43 gene.
  • the mutation may be a mutation of c.C1114T in the nucleotide sequence of SEQ ID NO: 2.
  • the mutation c.C1114T in the nucleotide sequence of SEQ ID NO: 2 is a mutation in which the cytidine (C), which is the 1114th nucleotide from the 5 'end of SEQ ID NO: 2 in the polynucleotide including the nucleotide sequence of SEQ ID NO: 2, Lt; / RTI >
  • the mutation may be the p.R372X mutation in the amino acid sequence of the TMEM43 protein.
  • the mutation may be a p.R372X mutation in the amino acid sequence of SEQ ID NO: 1.
  • the p.R372X mutation in the amino acid sequence of SEQ ID NO: 1 is a mutation in which the arginine (R), which is the 372nd amino acid from the N terminus of SEQ ID NO: 1, is deleted by the termination codon in the polypeptide comprising the amino acid sequence of SEQ ID NO: have.
  • a polynucleotide comprising a contiguous nucleotide sequence selected from the nucleotide sequence of the exon region of the TMEM43 gene or a complementary polynucleotide thereof for detecting the TMEM43 mutation is a polynucleotide comprising a single nucleotide sequence at the polynucleotide comprising the nucleotide sequence of SEQ ID NO: Or a polynucleotide capable of detecting the 1114th nucleotide from the 5'end of SEQ ID NO: 2.
  • the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 may be the same or complementary to the polynucleotide comprising the 5'-end to the 1114-th nucleotide of SEQ ID NO: 2, which is a single nucleotide mutation position. If one single-stranded polynucleotide is associated with the risk of developing a sensory neural deafness disorder, the polynucleotide complementary to the single-stranded polynucleotide may naturally be associated with the risk of developing a sensory neural deafness disorder.
  • the composition may comprise a single-stranded polynucleotide and / or a polynucleotide having a nucleotide sequence complementary thereto, which is associated with the risk of developing a sensory neural deafness disorder with one particular nucleotide sequence.
  • compositions for the diagnosis of sensory neuron deafness disorder comprising a polypeptide for specifically detecting a polypeptide in which some amino acids of TMEM43 have been lost to detect TMEM43 mutation.
  • the composition may include a polypeptide capable of detecting the amino acid deletion position in the TMEM43 protein.
  • the composition may include a polypeptide capable of detecting the 372nd amino acid from the N-terminus of SEQ ID NO: 1, which is an amino acid deletion position in a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • Amino acid mutations may be due to nonsense mutation mutations.
  • a nonsense mutation refers to a change in which one or more nucleotides are changed to termination codons, thereby no longer producing amino acids.
  • the polypeptide may specifically bind to the 372nd amino acid from the N-terminus of SEQ ID NO: 1, which is the position at which the amino acid terminates and disappears in the polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • the 372nd amino acid may be a polynucleotide encoding the 1114th nucleotide from the 5'end of SEQ ID NO: 2
  • the polypeptide may be a polynucleotide encoding the nucleotide 1114th from the 5'end of SEQ ID NO: 2
  • And may be one capable of detecting the resulting amino acid sequence.
  • the polypeptide may be an antibody or an antigen-binding fragment, and may be singular or plural.
  • the antibody may be one which is not only an entire antibody form but also contains a functional fragment of an antibody molecule.
  • the whole antibody is a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond.
  • a functional fragment of an antibody molecule means a fragment having an antigen-binding function.
  • the polypeptide may be prepared by immunocytochemistry and immunohistochemistry, radioimmunoassays, enzyme linked immunoabsorbent assay (ELISA), immunoblotting, Farr assay, immunoprecipitation, Aggregation, erythrocyte aggregation, an ascending method, an immunodiffusion method, a counter-current electrophoresis method, a single radical immunodiffusion method, an immunochromatography method, a protein chip and an immunofluorescence method. That is, a method capable of measuring the binding of an antigen to an antibody.
  • ELISA enzyme linked immunoabsorbent assay
  • kits for a sensory neuron deafness disorder comprising a polynucleotide comprising a contiguous nucleotide sequence selected from the nucleotide sequence of the exon region of the TMEM43 gene or a complementary polynucleotide thereof for detecting the TMEM43 mutation.
  • the kit may comprise a polynucleotide capable of detecting the 1114th nucleotide from the 5'end of SEQ ID NO: 2, which is a single nucleotide mutation position in a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2.
  • polynucleotide having the same or complementary sequence as the polynucleotide comprising the 5'-end to the 1114-th nucleotide of SEQ ID NO: 2, which is a single nucleotide mutation position in the polynucleotide including the nucleotide sequence of SEQ ID NO: 2 have.
  • the kit may, for example, comprise the polynucleotide and the constructs necessary for its particular use. And the reagent necessary for the method of use thereof together with the polynucleotide.
  • the kit may further comprise a known material required for hybridization of the polynucleotide with the nucleic acid.
  • the kit may be a kit for specifically amplifying the nucleotide sequence of the target region and diagnosing the sensory neuron deafness disorder of the individual through the presence or absence of the amplification product. Or a kit for hybridizing the polynucleotide or a probe derived therefrom with a nucleic acid in a sample and diagnosing the risk of developing an individual's sensory neural deafness disorder from the hybridization result.
  • the kit may further comprise a reagent, buffer, buffer, cofactor, and / or substrate necessary for hybridization of the nucleic acid.
  • a reagent necessary for PCR amplification for example, a buffer, a DNA polymerase, a DNA polymerase cofactor, and dNTPs.
  • the kit may further include instructions for use to amplify the target region, and may be produced from a number of separate packaging or compartments containing the reagent components described above.
  • the target region is amplified in the amplification reaction using the specific primer and the target region is not amplified in the amplification reaction using the non-specific primer, May include an explanation of the result determination, including an explanation of determining that an associated target region or variation is present and determining the risk of developing the sensory neural deafness disorder of the subject from the result.
  • Another aspect provides a diagnostic kit for a sensory neuron deafness disorder comprising a polypeptide for specifically detecting a polypeptide in which some amino acids of TMEM43 have been deleted to detect the TMEM43 mutation.
  • kits comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, one or more of which is suitable for the method of assaying a polypeptide for determining the level of expression of the 372nd amino acid- Other components or devices may be included.
  • the expression level of the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 is determined by measuring the presence and the expression level of the polypeptide or protein expressed in the gene and comparing the expression level of the polypeptide comprising the amino acid sequence of SEQ ID NO: Can be used to confirm the expression level of the gene or the amount of the protein.
  • the kit may optionally comprise a secondary antibody and a labeling substrate.
  • Another aspect provides a vector comprising a polynucleotide consisting of a nucleotide sequence encoding the p.R372X mutation of TMEM43.
  • the vector may comprise a polynucleotide consisting of a nucleotide sequence encoding the p.R372X mutation in the amino acid sequence of SEQ ID NO:
  • the vector means the carrier of the polynucleotide.
  • the polynucleotide may be in a vector that is a suitable expression system, for example, an expression vector.
  • a polynucleotide consisting of a nucleotide sequence encoding the p.R372X mutation in the amino acid sequence of SEQ ID NO: 1 may be operably linked to a promoter.
  • operably linked means a functional linkage between an array of nucleic acid expression control sequences, for example, an array of promoter, signal sequence, or transcription factor binding sites and other nucleic acid sequences, Regulate transcription and / or translation of other nucleic acid sequences.
  • Promoters that can be used in the expression vector are those capable of regulating the transcription of the nucleotide sequence by working in an animal cell such as a mammalian cell, such as a promoter derived from a mammalian virus and / or a promoter derived from the genome of a mammalian cell . ≪ / RTI > For example, cytomegalo virus (CMV) promoter, late adenovirus promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallothionein promoter, or Beta-actin promoter.
  • the vector may comprise an available polyadenylation sequence.
  • the backbone may be selected as necessary from various vectors suitable for expression of the polynucleotide.
  • pCMV6-Entry pHY92 vector, pRC CMV vector, pIRES2-EGFP vector, SV40 vector, papilomavirus vector, YIp5 vector, YCpl9 vector, Absteinvear virus vector, pMSG vector, pMAMneo- , PSECTag2B vector, or yT &
  • a vector for example, pCMV6-Entry, pHY92 vector, pRC CMV vector, pIRES2-EGFP vector, SV40 vector, papilomavirus vector, YIp5 vector, YCpl9 vector, Absteinvear virus vector, pMSG vector, pMAMneo- , PSECTag2B vector, or yT & A vector.
  • a polynucleotide consisting of a nucleotide sequence encoding the p.R372X mutation of TMEM43 inserted in the pCMV6-Entry vector may be produced by methods known in the art, such as position directed mutagenesis and polymerase chain reaction have.
  • Another aspect provides a sensory neural deafness disorder model comprising TMEM43 mutations.
  • the model may be a transformation having a TMEM43 mutation.
  • transformant means a transformed cell or a transformed animal into which a gene encoding one or more desired proteins has been introduced.
  • the transformed animal refers to an animal that continuously expresses TMEM43 having the p.R372X mutation.
  • the transformed animal may comprise a mutation of the nucleotide sequence of the exon region of the TMEM43 gene.
  • the transformed animal may be one which comprises the c.C1114T mutation in the nucleotide sequence of the TMEM43 gene.
  • the transformed animal may be an animal that constantly expresses TMEM43 with the p.R372X mutation integrated into the animal's chromosome by injecting the animal's gene encoding the TMEM43 with the p.R372X mutation into the embryo.
  • the gene coding for the TMEM43 having the p.R372X mutation may be a somatic cell or a germ cell of a transformed animal, or a genome of a somatic cell and a germ cell.
  • the transformed animal can be obtained by microinjecting the vector into fertilized or embryonic stem cells (ES cells) (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et et al., EMBO J., 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., et al., Virology, 52: 456 (1973) , Gene, 10: 87 (1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)), retroviral infection and gene bend al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)).
  • ES cells fertilized or embryonic stem cells
  • ES cells fertilized or embryonic stem cells
  • Ca phosphate precipitation method Graham, FL et et al., EMBO J., 1: 841 (1982)
  • the transgenic animal may be transiently transfected with a nucleic acid encoding a nucleic acid encoding a nucleic acid encoding a nucleic acid encoding a nucleic acid molecule selected from the group consisting of programmable nuclease such as zinc finger nuclease (ZFN), transcriptional activator-like effector nuclease (TALEN), CRISPR / Cas, or CRISPR / And the like.
  • ZFN zinc finger nuclease
  • TALEN transcriptional activator-like effector nuclease
  • CRISPR / Cas CRISPR / Cas
  • CRISPR / And the like programmable nuclease
  • the transformed animal is further provided with a step of delivering a polynucleotide consisting of a nucleotide sequence encoding the p.R372X mutation of the TMEM43 to a fertilized egg or embryonic stem cell followed by preparing a fertilized animal, Transplanting into the uterus of an animal, raising the animal so that the embryo is born as a baby, obtaining offspring to select whether expression of the p.R372X mutation of TMEM43 is to be performed.
  • the transformed animal may be a mouse, rat, cow, horse, pig, sheep, goat, dog, cat, or the like, including but not limited to various mammals other than humans.
  • the transformed cell refers to a cell that continuously expresses TMEM43 having the p.R372X mutation.
  • the cells may be somatic cells or germ cells, or somatic cells and germ cells.
  • the transfected cells may be prepared by a known method for introducing the vector into cells, and may be prepared by a suitable standard technique, for example, microinjection, calcium phosphate precipitation, electroporation, liposome -Mediated transfection, DEAE-dextran treatment, retroviral infection, and gene bombardment, and the like.
  • the vector of circular form can be introduced into a linear vector form by cutting with appropriate restriction enzymes.
  • the transformant since the transformant continuously expresses the TMEM43 having the p.R372X mutation, it may be one used for screening a therapeutic agent for hearing loss or a therapeutic method. Screening for the therapeutic agent for sensory neural damage is performed by adding a test substance to be analyzed to a transformant that continuously expresses TMEM43 having a p.R372X mutation, and further comprising the step of analyzing the degree of treatment or alleviation of sensory nerve impairment in the transformant And evaluating the quality of the image.
  • the addition of the test substance to be analyzed to the transformed cells or the transformed animal can be carried out by various methods known to those of ordinary skill in the art.
  • Another aspect includes identifying a nucleotide sequence of a genomic DNA isolated from an individual; And comparing the confirmed nucleotide sequence of the genomic DNA with a standard nucleotide sequence to identify the mutation of the genomic DNA.
  • the method comprises identifying the nucleotide sequence of the genomic DNA isolated from the subject.
  • the subject refers to an object for predicting the risk of developing sensory neural deafness.
  • the subject may be a subject suspected of having or developing a sensory neural deafness disorder.
  • the subject may be a vertebrate animal, a mammal, a human ( Homo sapiens ), a mouse, a rat, a cow, a horse, a pig, a sheep, a goat, a dog, a cat and the like.
  • the human being may be Asian or Korean. &Quot; Subject " and " patient " are used interchangeably herein.
  • the genomic DNA isolated from an individual in the method may be a genomic DNA isolated from a biological sample or a fragment thereof.
  • the biological sample may be a blood sample, tissue, urine, mucus, saliva, tear, plasma, serum, sputum, spinal fluid, pleural effusion, aspiration nipple, lymphatic fluid, airway fluid, Intracranial fluid, ascites fluid, cystic tumor fluid, positive fluid, or a combination thereof.
  • the biological sample may be one comprising a purely isolated nucleic acid, a crude nucleic acid, a cell lysate comprising a nucleic acid, or a cell free nucleic acid.
  • the method of separating the genomic DNA from the biological sample can be performed by a conventional nucleic acid separation method.
  • Identifying the nucleotide sequence of the genomic DNA in the above method means determining the nucleotide at the target region or each position in the chromosome.
  • the nucleotide sequencing method of a known nucleic acid can directly determine the nucleotide at the target region or at each position in the chromosome.
  • the nucleotide sequence determination method may include a ginger (or dideoxy) sequencing method or a germ-gilbert (chemical cleavage) method.
  • the nucleotide sequence of the target region can be determined by hybridizing the probe with the polynucleotide of interest and analyzing the result of hybridization.
  • the degree of hybridization can be confirmed, for example, by marking a detectable label on the target nucleic acid, detecting the hybridized target nucleic acid, or confirming it by an electrical method or the like.
  • a single base primer extension (SBE) method can be used.
  • the nucleotide sequence determination method may also include the next generation base sequencing.
  • next generation sequencing " (NGS) involves fragmenting a full-length genome in a chip-based and PCR-based paired end format, Sequencing. By next-generation nucleotide sequencing, a large amount of nucleotide sequence data can be generated for a sample to be analyzed within a short time.
  • the method may include fragmenting the isolated genomic DNA to an arbitrary size. Such fragmentation can be performed by methods known to those of ordinary skill in the art. For example, genomic DNA can be fragmented by using ultrasonic waves.
  • the method may include fragmenting the genomic DNA, and ligation of sequences for amplification at both ends of the fragmented genomic DNA.
  • a sequence for the amplification for example, a paired-end tag, a universal tag, or the like can be appropriately selected and performed by a person skilled in the art.
  • the method may comprise obtaining a separated genomic DNA and a hybridization product of the polynucleotide in the composition.
  • the method may be to obtain a hybridization product of a polynucleotide contained in the composition and a fragment of a genomic DNA having a nucleotide sequence of a target region targeted by the polynucleotide.
  • the hybridization can be accomplished by contacting the composition with isolated genomic DNA.
  • the hybridization can be carried out by known methods. For example, by incubating the genomic DNA isolated from the polynucleotide in a buffer known to be appropriate for the hybridization of the nucleic acid. Hybridization can be performed at an appropriate temperature.
  • Suitable temperatures for hybridization may be, for example, from about 40 ⁇ C to about 80 ⁇ C, from about 50 ⁇ C to about 75 ⁇ C, from about 60 ⁇ C to about 70 ⁇ C, or from about 62 ⁇ C to about 67 ⁇ C.
  • the hybridization temperature is not limited thereto, and can be appropriately selected according to the nucleotide sequence and the length of the polynucleotide contained in the composition.
  • the hybridization time can be, for example, from 1 hour to 12 hours (overnight).
  • the method may further include, after obtaining the hybridization product of the separated genomic DNA and the polynucleotide in the composition, separating the separated genomic DNA and the hybridization product of the polynucleotide before identifying the nucleotide sequence of the separated genomic DNA Step < / RTI >
  • the separation may be by using a moiety for separation or purification attached to the polynucleotide.
  • the separation or purification may be carried out by a substance or a magnetic field that specifically binds to the moiety.
  • the method comprises using the isolated hybridized product or separated genomic DNA as a template, sequencing amplification sequences at both ends of the template, and using a universal primer complementary to the sequence for amplification as a primer And amplifying the isolated hybridized product or the separated genomic DNA by PCR using the hybridization product.
  • the amplified product can be used to confirm the nucleotide sequence.
  • the method comprises comparing the identified nucleotide sequence of the genomic DNA with a standard nucleotide sequence to identify the variation of the genomic DNA.
  • the term " reference neucleotide sequence" may be a human genomic sequence that does not contain a mutation that is referenced for mutation confirmation.
  • the standard nucleotide sequence may be the nucleotide sequence of the reference genome, for example, the nucleotide sequence of a human gene, specifically NCBI37.1 or UCSC hg19 (GRCh37), published in the database of the Institute of Biotechnology Information, .
  • the comparison between the nucleotide sequence of the genomic DNA and the standard nucleotide sequence can be performed using various known sequence comparative analysis programs such as Maq, Bowtie, SOAP and GSNAP.
  • the method may further comprise, when the mutation of the genomic DNA is confirmed, determining that the subject belongs to a high-risk group having a sensory neural deafness disorder. That is, the individual can be diagnosed as having sensory neural deafness.
  • the risk group refers to a group predicted or diagnosed as having an increased probability of developing a sensory neural deafness disorder compared to a group having a standard nucleotide sequence or a normal group.
  • Another aspect is the use of a polynucleotide comprising a contiguous nucleotide sequence selected from the nucleotide sequence of the exon region of the TMEM43 gene to detect a TMEM43 mutation, or a polynucleotide comprising its complementary polynucleotide, ≪ / RTI >
  • the prognosis for wahoo implantation in patients with sensory nerve impairment means predicting whether speech can be understood during wah-wedge surgery or whether speech can be better understood after wahoo implantation. For example, it may be to predict whether the auditory brainstem response will normally appear.
  • the patient with sensory neuralgic hearing loss having the above mutation has abnormality in the supporting cells around the inner or inner hair cell, and there is no problem in the conduction of the auditory nerve. Therefore, the prognosis of the wah operation can be expected to be positive.
  • Another aspect includes identifying a nucleotide sequence of a genomic DNA isolated from an individual; And comparing the identified nucleotide sequence of the genomic DNA with a standard nucleotide sequence to identify the mutation of the genomic DNA.
  • identifying a nucleotide sequence of a genomic DNA isolated from an individual and comparing the identified nucleotide sequence of the genomic DNA with a standard nucleotide sequence to identify the mutation of the genomic DNA.
  • Identifying a nucleotide sequence of the genomic DNA isolated from the subject; And comparing the confirmed nucleotide sequence of the genomic DNA with a standard nucleotide sequence to confirm the mutation of the genomic DNA is as described above.
  • Providing information for predicting the prognosis of the wah-graft operation in the patients with sensory-nerve impairment means predicting whether the speech will be understood during the wah-graft operation or whether the speech will be better understood after the wah-graft operation . For example, it may be to predict whether the auditory brainstem response will normally appear.
  • the mutation of the TMEM43 gene can be analyzed with high sensitivity and accuracy, and the sensory nerve impairment can be efficiently diagnosed based on the analysis result.
  • FIGS. 1A and 1B show the process of confirming the genealogy and the TMEM43 mutation to which members of the cohort SB162 belong.
  • Figure 2 shows the result of confirming the organ in which TMEM43 is expressed in mouse.
  • FIG. 3 shows the results of confirming the expression of TMEM43 in the inner ear of 4 to 5 days old and 20 days old mice.
  • FIG. 4 shows the results of confirming the expression pattern of TMEM43 in the inner ear of 4-day, 15-day, and 20-day-old mice.
  • Figure 5 shows the process for constructing a transformed animal having the p.R372X mutation of TMEM43.
  • Auditorin means TMEM43.
  • Figure 6 shows the results for the 2, 7, and 13 months of TMEM43 + / + and TMEM43 + / tm1Cby An image of a transcriptional micrograph showing the inner border cell and the border cell in the reticular lamina of mouse hair cells.
  • green and pale green represent inner boundary cells
  • orange and yellow represent boundary cells, respectively.
  • Figure 7 shows the results of the 6 and 9 month old TMEM43 + / + and TMEM43 + / tm1Cby And the average ABR threshold for 4, 8, 16, and 32 kHz in the mouse. Auditorin means TMEM43.
  • 8A to 8H show the results of confirming the function of TMEM43 using cell patch clamp technology in CHO-K1 cells expressing TMEM43.
  • Example 1 From patients with sensory nerve impairment TMEM43 p. R372X Detection of mutation and confirmation of sensory neural deafness in animal model with p.R372X mutation of TMEM43
  • SB162 a family tree was isolated by autosomal dominant inheritance method of adult onset type progressive auditory neuropathy based on standardized hearing test or family history data. Compared with normal subjects (290), subjects (291, 284, and 304) who were affected by auditory neuropathy decreased their susceptibility to sound at Pure Tone Average (PTA) (DPOAE) or cochlear microphonic potential (CM), which has a complete absence of Auditory Brainstem Responses (ABRs) and is impaired in the Speech Discrimination Score (SDS) It was normal.
  • PTA Pure Tone Average
  • CM cochlear microphonic potential
  • adult auditory neuropathy In a total of 1,100 households, 780 households with hearing impairments, 15 families selected adult auditory neuropathy households. In the case of adult auditory neuropathy, the age at onset was secondarily selected for families with a language acquisition period. Based on clinical features, electrophysiologic findings, and genetic diagnosis in the candidate gene pool, adult auditory neuropathy was then classified into three types:
  • Type 1 If known to be caused by a known hearing loss gene (eg DIAPH3)
  • E-ABR electro-evoked auditory brainstem response
  • Target exome sequencing was performed on the 3p25-26 region from the linkage analysis for 4 subjects (SB162-284, 289, 290, 291) and the candidate variants were listed.
  • a mutation with a minor allele frequency of less than 1% was identified in ESP6500 and 1000G.
  • the dbSNP which is not a flagged dbSNP, is filtered, leaving a variation that matches the genetic pattern.
  • 622 species of normal control were used to eliminate Korean specific mutations. Genome level log2 ratios for chromosome 3 were examined.
  • Sequencing leads were aligned to the human reference genome (hg19) using BWA v 0.7.5 and SAMTOOLS v 0.1.18, and sorted, indexed, rearranged, and duplicated using CATK v 2.4-7 and Picard v 1.93 .
  • the mutation was named using the GATK Unified Genotyper and the mutation was reaffirmed. Variations were annotated using ANNOVAR. Thereafter, the final candidate variation of the household was confirmed by Sanger sequencing.
  • Candidate mutations were filtered by the variation of the coding region (synonymous variant) and the noncoding region.
  • FIGS. 1A and 1B show the process of confirming the genealogy and the TMEM43 mutation to which members of the cohort SB162 belong. After the filtering process based on the dbSNP database, allele frequency, and genetic pattern, and mutations of R372X in TMEM43 were detected as heterozygotes, the number of mutations identified in the process of selecting mutations of genes is shown in Table 3 below.
  • the p.R372X mutation is presumed to be inherited as autosomal dominant. In addition, the p.R372X mutation was not detected in the chromosomes of the Korean control group with normal hearing.
  • 129Sv / Ev mice were anesthetized with pentobarbital sodium (50 mg / kg).
  • pentobarbital sodium 50 mg / kg.
  • TRIZOL ® Gaithersburg, MD, USA
  • RNeasy kits using (Qiagen, Valencia, USA), the entire cochlea (cochlea) (P1 ), Heart (P120), eyes (P42), brain (P28), kidney (P28), and liver (P28).
  • cDNA was synthesized using oligo dT primer and extracted RNA. Polymerase chain reaction (PCR) was performed using the synthesized cDNA and a specific primer set specific for TMEM43 and Gapdh.
  • the PCR conditions were as follows: 95 ° C for 2 minutes, 95 20 sec at 55 ° C, 10 sec at 55 ° C, and 35 min at 70 ° C and 5 min at 72 ° C.
  • the amplification product (15 ⁇ l) was electrophoresed on 2% agarose gel and visualized with ethidium bromide.
  • TMEM43 forward primer 5'-CTTCCTGGAACGGCTGAG-3 '(SEQ ID NO: 3) and TMEM43 reverse primer 5'-CACCAGCCTTCCTTCATTCT-3' (SEQ ID NO: 4)
  • Gapdh forward Primer 5'-ACCACAGTCCATGCCATCAC-3 ')
  • Gapdh reverse primer 5'-CACCACCCTGTTGCTGTAGCC-3' (SEQ ID NO: 6).
  • FIG. 2 shows the result of confirming the organ in which TMEM43 is expressed in mouse. As shown in FIG. 2, it can be seen that TMEM43 is expressed in the inner ear cochlea.
  • NH 2 mouse TMEM43 - Rabbit polyclonal anti-serum (AbClon) that instructs the terminal was produced.
  • Peptide sequences are as NH 2 -SRKEHVKVTSE-COOH (SEQ ID NO: 7).
  • the primary antibodies were rabbit anti-TMEM43 monoclonal antibody (1: 500, Abcam, Ab184164), Rabbit anti-TMEM43 polyclonal antibody (1: 100, Novus, Cat # NBP1-84132, Littleton, USA 80120) (1: 500, Abcam, ab125096), mouse anti-calretinin monoclonal antibody (1: 500, Millipore, MAB1568), gut polychlorinated anti-Na + , K + ATPase- ⁇ 3 antibody (NKA, 1: 250, Santa Cruz, SC-16052) and mouse monoclonal anti-CTBP2 antibody (1: 500, BD Transduction Laboratories, 612044) were used. Secondary antibodies were diluted 1: 1000 with donkey or life technologies.
  • the tissue sample was mounted on a glass slide using a Fluorsave reagent (Calbiochem, 345789), covered with a coverslip, and a microscope sample was made. Images were obtained from a microscope sample using an epifluorescence microscope and a confocal laser scanning microscope (LSM710, Zeiss).
  • FIG. 3 shows the results of confirming the expression of TMEM43 in the inner ear of 4 to 5 days old and 20 days old mice. As shown in FIG. 3, it can be seen that TMEM43 is expressed in supporting cells around the inner hair cells.
  • FIG. 4 shows the results of confirming the expression pattern of TMEM43 in the inner ear of 4-day, 15-day, and 20-day-old mice. As shown in Fig. 4, TMEM43 in mouse is expressed in inner ear, and it is constantly expressed in supporting cells around inner ear embryonic cell even when time passes. In addition, the expression pattern of TMEM43 was consistent with the pattern of spontaneous activity (not shown).
  • a mouse model of TMEM43 -R372X knock-in (C57BL / 6J; 129S-TMEM43 tm1Cby ) mouse model was constructed using a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) method.
  • FIG. 5 shows the process for constructing a transformed animal having the p.R372X mutation of TMEM43.
  • arginine (R) was replaced with a stop codon at position 372 of the TMEM43 protein (gene ID: NM_028766).
  • gRNA was constructed. Two gRNA candidates located close to the mutation site ( TMEM43 gRNA1: 5'- TTAGAGCAGCCCACAGCGGT CGG- 3 '(SEQ ID NO: 8); TMEM43 gRNA2: 5'-CCGATTAGAGCAGCCCACAG CGG- 3 '(SEQ ID NO: 9)) was evaluated.
  • CCG represents a PAM (Protospacer Adjacent Motif) sequence.
  • SURVEYOR assay was performed using the SURVEYOR Mutation Detection Kit (IDT) according to the manufacturer's instructions. Based on the identified gRNAs, a single stranded oligodeoxynucleotide donor (ssODN) was designed and synthesized. The 160 bp donor DNA contains two homologous arms flanking the mutation (C- > T) introduced in exon 12 corresponding to the 1114 position of the TMEM43 gene. In addition, additional mutations corresponding to the second termination codon were introduced to prevent the repaired genome from being retargeted by the Cas9 complex.
  • IDT SURVEYOR Mutation Detection Kit
  • C57BL / 6J embryos Sixty-two C57BL / 6J embryos were injected via the cytoplasm with a CRISPR cocktail containing ssODN donor, gRNA transcript TMEM43, and Cas9 mRNA. Of the 62 embryos, 49 embryos were selected for screening and transplanted into two CD1 mice. All females were pregnant and gave birth to nine litters of F0. Female F0 was crossed with wild-type male C57BL / 6J mice, F1 was obtained, and germline transmission of germ cells was confirmed. For all mice, the presence or absence of a point mutation at the target site was analyzed by PCR and sequencing.
  • genomic DNA was extracted from the tail of about 1 mm to about 2 mm using DNeasy ® blood & tissue kit (Qiagen, Hilden, Germany). Using 5 ⁇ l of genomic DNA, 25 mM MgCl 2 , 2 mM of dNTPs, 10 pM of primer, and 0.02 U of TOYOBO KOD Hot Start DNA polymerase (Invitrogen / Life Technologies, Billerica, Massachusetts, USA) The PCR conditions were as follows: 95 ° C for 2 minutes, followed by 95 ° C for 20 seconds, 59 ° C for 10 seconds, and 72 ° C for 10 seconds and 72 ° C for 10 minutes.
  • primer sequences were as follows: a forward primer 5'-ccacagTGGACTGGTTTCCT-3 '(SEQ ID NO: 10), and a reverse primer 5'-GGCTTCACTCCAGCTTTTTG-3' 11).
  • the size of the amplified product by the primer sequence is about 213 bp.
  • the amplified product was confirmed by Sanger sequencing (Macrogen Inc., Seoul, KOR).
  • tissue sample was prepared using osmium tetroxide - thiocarbohydrazide method and observed with a scanning electron microscope.
  • the tissue samples were then dehydrated in an ethanol solution with a concentration gradient, dried and platinum coated using a sputter coater (E1030; Hitachi, Tokyo, Japan).
  • the surface of the Corti organ was captured with a cold field emission SEM (SU8220, Hitachi, Tokyo, Japan) operating at 10 or 15 kV.
  • Microscopic images were obtained using ImageJ software (National Institutes of Health, http://rsbweb.nih.gov/ij/).
  • Figure 6 shows the results for the 2, 7, and 13 months of TMEM43 + / + and TMEM43 + / tm1Cby
  • green and pale green represent inner boundary cells
  • orange and yellow represent boundary cells, respectively.
  • TMEM43 + / tm1Cby The area of the inner border cells and the border cells of the mouse is decreased as compared with that of the inner border cells and the border cells of wild type mice. It is expected that as the inner border cells and border cells shrink and decrease, the auditory nerve transposition is suppressed.
  • ABR was performed in an anechoic room. Mice were treated with 0.1 mg levomepromazin chlorhydrate and about 3.5 mg ketamine chlorhydrate was intravenously administered to 15 g mice by intraperitoneal injection. The ABR collected and analyzed the response to a short tone burst calibrated in the range of 4 to 32 kHz. Electroencephalograms were recorded via stainless steel electrodes under the vertices and ipsilateral mastoids using a standard digital averaging system. At all sound levels, 500 responses to tone bursts were synchronous averaging. ABRs greater than the threshold consisted of waves of regular intervals (I to IV).
  • the tone burst level varies from 10 dB to 120 dB SPL peak-equivalent, while the ABR threshold was obtained at the minimum stimulation level required to generate at least one repeatable wave IV 40.3 mV.
  • Figure 7 shows the mean ABR threshold for 4, 8, 16, and 32 kHz in TMEM43 + / + and TMEM43 + / tm1Cby mice at 6 and 9 months of age. As shown in Fig. 7, TMEM43 + / tm1Cby The mouse shows that the ABR threshold is higher than the wild type ABR threshold.
  • TMEM43 The function of TMEM43 was confirmed using a cell patch-clamp technique.
  • Cell patch clamp technology is known as a method of measuring electrical signals.
  • An electrode in a glass micropipette can measure the electrical signal from one cell.
  • the characteristic of the ion channel developed in the cell can be known.
  • human TMEM43 was expressed in Chinese hamster ovary (CHO-K1) cells and membrane current was measured under voltage clamp.
  • FIG. 8 shows the results of confirming the function of TMEM43 using cell patch clamp technology in CHO-K1 cells expressing TMEM43.
  • CHO-K1 cells in which TMEM43 protein is almost not expressed do not pass an electric signal, but CHO-K1 cells expressing TMEM43 protein transmit electric signals (Fig. 8A).

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Abstract

La présente invention concerne des marqueurs destinés au diagnostic de la perte d'audition neurosensorielle et l'utilisation de ceux-ci. L'invention concerne notamment : une composition de diagnostic de la perte d'audition neurosensorielle contenant un polynucléotide, lequel comprend une séquence nucléotidique continue qui est choisie parmi des séquences nucléotidiques dans une région exon du gène TMEM43, afin de permettre la détection de mutations du gène TMEM43, ou un polynucléotide complémentaire de celui-ci; et un procédé de détection de la mutation d'ADN génomique afin de fournir des informations pour le diagnostic. Selon un mode de réalisation de l'invention, il est possible d'utiliser un kit ou une composition pour analyser la mutation du gène TMEM43 avec une sensibilité et une précision élevées, et de diagnostiquer efficacement la perte d'audition neurosensorielle sur la base des résultats d'une telle analyse.
PCT/KR2018/012442 2017-10-19 2018-10-19 Marqueur de tmem43 destiné au diagnostic de la perte d'audition neurosensorielle et son utilisation Ceased WO2019078684A2 (fr)

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WO2022032151A1 (fr) * 2020-08-07 2022-02-10 University Of Miami Traitement de la surdité de perception

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KR102326582B1 (ko) 2020-05-15 2021-11-16 사회복지법인 삼성생명공익재단 청각장애의 진단용 마커 및 그의 용도
JP7699789B2 (ja) * 2021-04-30 2025-06-30 ソウル ナショナル ユニバーシティ ホスピタル 感音性難聴の原因特異的治療効果を予測する方法及びそのために使用される診断キット
KR20240175764A (ko) * 2023-06-13 2024-12-23 서울대학교병원 난청 예측 또는 진단용 조성물

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022032151A1 (fr) * 2020-08-07 2022-02-10 University Of Miami Traitement de la surdité de perception

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