KR102634424B1 - Novel KCNQ4 protein variant and use thereof - Google Patents

Abstract

It relates to a novel KCNQ4 protein variant, a composition and kit for diagnosing potassium channelopathy using the same, and a method of providing information using the same.
According to a composition for diagnosing potassium channelopathy, a kit, and a method for providing information for diagnosing potassium channelopathy using the same, the occurrence and prognosis of potassium channelopathy can be determined objectively, quickly, with high accuracy and specificity. It can be used for road detection.

Description

Novel KCNQ4 protein variant and use thereof {Novel KCNQ4 protein variant and use thereof}

It relates to a novel KCNQ4 protein variant, a composition and kit for diagnosing potassium channelopathy using the same, and a method of providing information using the same.

Hearing impairment is the most common sensory organ disorder in humans. Approximately 1 in 1,000 people (0.1%) show hearing impairment greater than severe hearing loss at birth (congenital), and an additional 1 in 1,000 people become hearing impaired during childhood before becoming an adult. (acquired) occurs. As you age, the probability of hearing loss due to various causes such as ototoxic drugs, accidents, and pre-senile hearing loss increases. Compared to the population, people with severe hearing loss of 65 dBHL or more account for 0.3% of those aged 30 to 50, and 0.3% of those aged 60 to 70. In the age group, it increases to 2.3%. Recently, with the advent of effective vaccinations, infectious diseases such as influenza, measles, and mumps have decreased, and due to active enlightenment and treatment, inner ear damage due to ototoxic drugs or accidents has also decreased, resulting in a relatively increased incidence of hereditary hearing loss.

Since the inner ear has a very complex structure and various cells are designed to perform organic functions, it is expected that the genetic diseases that cause hearing loss will be very diverse. Recent developments in molecular biology research have brought about great academic progress in uncovering the genes that cause hereditary hearing loss, and have led to an understanding of the mechanism by which hearing loss occurs, further reaching the stage of clinical application to find a cure.

Approximately one-third of hereditary hearing loss is syndromic, and the remaining two-thirds are nonsyndromic. Since syndromic hearing loss is easily classified by characteristic clinical symptoms, it is possible to collect many patients with the same cause and trace the causative gene. However, in the case of nonsyndromic hearing loss, which only shows hearing loss without other symptoms, the only way to find the causative gene is to analyze a large pedigree with many hearing loss patients.

Meanwhile, KCNQ4, a voltage-gated K ⁺ channel that plays an important role in the process of hearing sounds, is mainly expressed in the hair cells of the cochlea of the inner ear and acts as a regulator of K ⁺ ions in the cochlea. It plays a key role in maintaining homeostasis. When a mutation occurs in the KCNQ4 gene, non-syndromic progressive hearing loss (deafness nonsyndromic autosomal dominant 2, DFNA2) occurs. This hearing loss is progressive and causes moderate hearing loss. show opinions. Despite many studies attempted to prevent and treat hearing loss, a clear molecular mechanism and prevention and treatment plan for hearing loss have not yet been proposed.

Under this background, the present inventors made efforts to more effectively diagnose non-syndromic hearing loss and suggest treatment methods. As a result, they discovered a novel KCNQ4 mutation and confirmed that non-syndromic hearing loss can be effectively diagnosed through this, leading to the present invention. was completed.

One aspect provides a composition for diagnosing potassium channelopathy, comprising an agent for detecting a novel KCNQ4 protein variant, a fragment thereof, or a polynucleotide encoding the protein variant.

Another aspect provides a kit for diagnosing cardiovascular comorbidities in psoriasis patients comprising the diagnostic composition.

Another aspect provides a method of providing information for diagnosis or treatment of potassium channelopathy, comprising detecting or measuring the expression level of a KCNQ4 protein variant, fragment thereof, or polynucleotide encoding the protein variant.

Another aspect provides a composition for screening potassium channel activity inhibitors, comprising transformants expressing KCNQ4 wild-type protein and KCNQ4 protein variants.

Another aspect provides a method for screening potassium channel activity inhibitors using the transformant.

One aspect is a composition for diagnosing potassium channelopathy, comprising an agent for detecting a KCNQ4 (Voltage-Gated Potassium Channel Subunit Kv7.4) protein variant, a fragment thereof, or a polynucleotide encoding the protein variant, KCNQ4 protein variants consist of a) a variant in which alanine and aspartic acid, the 271st and 272nd amino acids, are deleted, b) a variant in which the 331st amino acid, arginine, is substituted with glutamine, and c) a variant in which the 319th amino acid, glycine, is substituted with aspartic acid. To provide a composition, which is one or more selected from the group.

As used herein, the term "KCNQ4 (Voltage-Gated Potassium Channel Subunit Kv7.4)" is a protein that constitutes a potassium channel present at the base of the hair cells of the inner ear, and maintains electrophysiological stability within the hair cells through the recycling of potassium. It is known to play a very important function in sound transmission.

When a mutation occurs in the KCNQ4 gene, calcium ions are not discharged from the hair cells due to loss of function of the potassium channel, and a state of depolarization within the hair cells is continuously maintained, resulting in gradual hearing loss due to cellular fatigue and potassium toxicity. This is known to occur.

In the case of the G319D variant, which is a novel KCNQ4 variant of the present invention, unlike other previously known mutations, non-syndromic hearing loss occurs due to a channel activity enhancement mechanism in which potassium channels are overactivated and calcium ions in hair cells leak excessively outward. It was confirmed that it was possible.

The KCNQ4 protein may include the amino acid sequence of SEQ ID NO: 1, and may specifically consist of the amino acid sequence of SEQ ID NO: 1.

The KCNQ4 protein variant (A271_D272del) in which the 271st and 272nd amino acids alanine and aspartic acid are deleted may include the amino acid sequence of SEQ ID NO: 3, and the KCNQ4 protein variant in which the 331st amino acid arginine is replaced with glutamine (R331Q ) may include the amino acid sequence of SEQ ID NO: 4, and the substituted KCNQ4 protein variant (G319D) in which the 319th amino acid, glycine, is replaced with aspartic acid may include the amino acid sequence of SEQ ID NO: 5.

The polynucleotide encoding the KCNQ4 protein variant is a) a mutation in which the 811th to 816th bases are deleted, b) a mutation in which the 992nd base, G, is replaced with A, and c) the 956th base, G, is replaced with A. It may include one or more selected from the group consisting of mutations. Specifically, a) the mutation in which the 811th to 816th bases are deleted encodes a mutant in which the 271st and 272nd amino acids, alanine and aspartic acid, are deleted, and b) the mutation in which the 992nd base, G, is replaced with A is the 331st amino acid. It encodes a variant in which arginine is substituted with glutamine, and c) the variant in which G, the 956th base, is substituted with A, may encode a variant in which glycine, the 319th amino acid, is substituted with aspartic acid.

The polynucleotide may include a polynucleotide fragment containing the mutation site of KCNQ4.

The polynucleotide encoding the KCNQ4 protein may include the base sequence of SEQ ID NO: 2, and may specifically consist of the base sequence of SEQ ID NO: 2.

As used herein, the term "potassium channelopathy" refers to a disease caused by dysfunction of potassium channels located in the membranes of various cells and organelles, specifically KCNQ4 protein or potassium channels composed of the above proteins. It may be caused by a malfunction of the KCNQ4 protein, and the malfunction of the KCNQ4 protein may be caused by a mutation in the KCNQ4 gene or a KCNQ4 protein variant.

As used herein, the term 'dysfunction' refers to a decrease, disappearance, or excessive increase in the normal biological activity of a protein in vivo. Therefore, dysfunction includes loss-of-function or gain-of-function. The above functional abnormalities may be due to abnormalities in gene transcription activity, abnormalities in translation, abnormalities in RNA processing, abnormalities in protein stability and three-dimensional structure, abnormalities in movement and activity, or a combination thereof.

If there is a malfunction in the KCNQ4 protein, a voltage gated potassium channel, the passage of potassium ions according to the action voltage is not normal, and the potassium ions introduced by stimulation cannot flow out of the cell or flow out excessively. As a result, the star quality of the potassium ion collapses. Therefore, potassium channelopathies that can be diagnosed with the composition of the present invention include all diseases in which potassium ion homeostasis is not maintained due to dysfunction of the KCNQ4 protein, such as neuromyotonia and episodic ataxia. Ataxia), hypokalaemic periodic paralysis, epilepsy, and non-syndromic deafness. Specifically, it may be non-syndromic hearing loss.

In this specification, the term "non-syndromic deafness" refers to a disease that shows only hearing loss due to inner ear dysfunction without other symptoms, and is used in the same sense as "non-syndromic sensorineural hearing loss." Non-syndromic hearing loss accounts for about two-thirds of hereditary hearing loss, is usually caused by a single gene abnormality, and shows several inherited forms. Depending on the locus of the gene, it is named as autosomal dominant (deafness nonsyndromic autosomal dominant (DFNA), autosomal recessive (DFNB), and sex chromosome genetic (DFN).

The composition may be capable of measuring the expression level of a KCNQ4 protein variant, a fragment thereof, or a polynucleotide encoding it. Specifically, the composition compares the expression level of the KCNQ4 protein variant or the polynucleotide encoding it with the expression level of the control group (KCNQ4 protein wild type, housekeeping protein, etc.), and determines whether the KCNQ4 protein variant is a heterozygous variant or mutation. It may be possible to determine whether it is a cognitive, homozygous variant, or mutation. The heterozygous mutation refers to a case where there is a mutation only on one side of a pair of homologous chromosomes, and the homozygous mutation refers to a case where there is a mutation on both sides.

Therefore, the composition can confirm whether the KCNQ4 protein variant is a heterozygous mutation. By using the composition, the KCNQ4 protein heterozygous variant in which glycine, the 319th amino acid, is substituted with aspartic acid is detected, resulting in hyperactivation of the potassium channel. Onset potassium channelopathy or non-syndromic hearing loss can be diagnosed.

As used herein, the term “detection or expression level measurement” refers to measuring the expression or expression level of a specific protein (peptide) or fragment thereof, or a gene encoding the protein. The expression level of the protein or fragment thereof may be a relative amount or an absolute amount of the protein or fragment thereof.

Measuring the expression level of the protein or fragment thereof may mean measuring the amount of the protein or fragment thereof. The expression level of the gene may be the expression level of mRNA encoding the proteins. The expression level of mRNA may be a relative amount or an absolute amount of mRNA. Measuring the expression level of the gene may mean measuring the amount of mRNA.

Methods for detecting or measuring the expression level of the protein include Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radical immunodiffusion, and octeroni immunoassay. Ouchterlony immunodiffusion, rocket immunoeletrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography It may be immunochromatography, fluorescence activated cell sorter analysis (FACS), or protein chip technology.

Methods for detecting or measuring the expression level of the mRNA or gene include reverse transcriptase polymerization reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), and real time quantitative RT-PCR. ), quantitative RT-PCR, RNase protection method, Northern blotting, or DNA chip technology.

As used herein, the term "agent for detecting or measuring the expression level" refers to a molecule that can be used to detect or confirm the expression level of a specific protein or the gene encoding it, and specifically detects and detects the protein or the gene. /Or it may contain an agent capable of amplification.

As used herein, the term “agent capable of detecting a specific gene or protein” refers to an agent that can specifically bind to and recognize the specific gene or protein, or detect and amplify the specific gene or protein. “Agent capable of amplifying a protein” refers to an agent that can increase the number of a specific gene or protein by repeating its replication, for example, a primer that can specifically amplify a polynucleotide containing the gene Alternatively, it may mean a probe that can bind specifically.

The agent may be an antibody, an antigen-binding fragment thereof, or an aptamer that specifically binds to the protein variant or fragment thereof. The term “antibody” may be used interchangeably with the term “immunoglobulin”. The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be a full-length antibody. The antigen-binding fragment refers to a polypeptide containing an antigen-binding site. The antigen binding fragment may be a single-domain antibody, Fab, F(ab'), F(ab') ₂ or Fv. The antibody or antigen-binding fragment may be attached to a solid support. The solid support is, for example, the surface of a metal chip, plate, or well. The aptamer may be a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) or peptide that specifically binds to the protein or fragment thereof.

The agent may be a nucleic acid containing a polynucleotide identical to or complementary to a polynucleotide encoding the protein variant or fragment thereof. The nucleic acid may be a primer, probe, or antisense oligonucleotide. The primer, probe, or antisense oligonucleotide may be labeled at its end or inside with a fluorescent substance, chemiluminescent substance, or radioactive isotope.

As used herein, the term "primer" refers to a nucleic acid sequence having a free 3' hydroxyl group, which can form base pairs with a template that is complementary to a specific base sequence and copies the template strand. refers to a nucleic acid sequence that serves as a starting point for. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature. For example, by performing PCR amplification using sense and antisense primers with 7 to 50 nucleotide sequences as specific primers for the mRNA of the gene encoding the biomarker of the present invention, the production amount of the desired product is measured to treat psoriasis. The likelihood of developing cardiovascular comorbidities in affected individuals can be predicted. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art. The primers are 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80. , may have 20 to 50, or 20 to 30 nt.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA that can specifically bind to a target nucleic acid, for example, mRNA, and to determine the presence, content, and expression level of a specific mRNA. It may be labeled so that it can be used. Probes may be manufactured in the form of oligonucleotide probes, single strand DNA probes, double strand DNA probes, RNA probes, etc. For example, hybridization is performed using a probe having a nucleic acid sequence complementary to the mRNA of the gene encoding the biomarker of the present invention, and the expression level of mRNA is measured through the degree of hybridization, thereby detecting cardiovascular comorbidities in individuals with psoriasis. The likelihood of an outbreak can be predicted. Selection of appropriate probes and hybridization conditions can be appropriately selected according to techniques known in the art. The probes are 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80. , may have 20 to 50, or 20 to 30 nt.

Additionally, primers or probes can be chemically synthesized using phosphoramidite solid support synthesis or other well-known methods. Additionally, these nucleic acid sequences can be modified through various methods known in the art. Examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologs, or modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate). etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.). Additionally, primers or probes may be modified using labels that can directly or indirectly provide a detectable signal. Examples of such labels may include radioactive isotopes, fluorescent molecules, or biotin.

As used herein, the term “diagnosis” refers to determining whether potassium channelopathy has already developed in a specific individual.

In this specification, the term "prediction" refers to determining whether a specific individual is likely to develop potassium channelopathy, and if so, whether the likelihood of developing potassium channelopathy is relatively high compared to an unspecified number of people. it means.

Another aspect is to provide a kit for diagnosing potassium channelopathy, including the composition for diagnosing potassium channelopathy. The same parts as described above also apply to the kit.

The kit has the effect of diagnosing or predicting the possibility of onset and prognosis of potassium channelopathy by detecting or measuring the expression level of the KCNQ4 protein variant, its fragment, or polynucleotide encoding it.

The kit may further include samples necessary to predict the onset of potassium channelopathy. The kit may include a solid support, a substrate for immunological detection of an antibody or antigen-binding fragment, a suitable buffer solution, a chromogenic enzyme, a secondary antibody labeled with a fluorescent substance, or a chromogenic substrate. The kit may include polymerase, buffer, nucleic acid, coenzyme, fluorescent substance, or a combination thereof for nucleic acid detection. The polymerase is, for example, Taq polymerase.

Specifically, the kit may be a PCR kit, RT-PCR kit, qRT-PCR kit, DNA chip kit, Western blot kit, or ELISA kit.

The PCR kit may be a kit containing essential elements required to perform PCR. A PCR kit, in addition to polynucleotides, primers or probes specific for the genes or methylation regions, contains test tubes or other suitable containers, reaction buffer (pH and magnesium concentration vary), deoxynucleotides (dNTPs), Taq-polymers, etc. It may include enzymes such as enzymes and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, and sterilized water.

The RT-PCR kit may be a kit containing the essential elements required to perform RT-PCR, and the RT-PCR kit may include a test tube or other appropriate container in addition to each primer specific for the genes or methylation regions. , reaction buffer (pH and magnesium concentration vary), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water. ), sterilized water, etc.

The DNA chip kit may be a kit containing the essential elements needed to perform the DNA chip, and the DNA chip kit includes a substrate to which a specific polynucleotide, primer, or probe for the genes or methylation regions is attached, The substrate may include a nucleic acid corresponding to a quantitative control gene or a fragment thereof.

Another aspect is providing information for diagnosis or treatment of potassium channelopathy, comprising detecting or measuring the expression level of a KCNQ4 protein variant, fragment thereof, or polynucleotide encoding the protein variant from a biological sample isolated from an individual. As a method, the KCNQ4 protein variants include a) a variant in which the 271st and 272nd amino acids alanine and aspartic acid are deleted, b) a variant in which the 331st amino acid arginine is substituted with glutamine, and c) the 319th amino acid glycine is substituted with aspartic acid. To provide a method, which is at least one selected from the group consisting of variants. The same parts as described above also apply to the above method.

As used herein, the term “individual” refers to any organism that develops or is likely to develop potassium channelopathy, and specific examples include mammals, such as humans, cattle, horses, pigs, dogs, sheep, goats, Or it could be a cat.

The biological sample refers to a sample obtained from the individual. The biological sample may be, for example, blood, plasma, serum, bone marrow fluid, lymph fluid, saliva, tear fluid, mucosal fluid, amniotic fluid, or a combination thereof. The biological sample includes a protein sample or a nucleic acid sample obtained from the sample, and the nucleic acid sample may include DNA, mRNA, or cDNA synthesized from mRNA.

The step of detecting or measuring the expression level may include incubating the biological sample with an antibody, antigen-binding fragment thereof, or aptamer that specifically binds to the protein variant or fragment thereof. The measuring step is performed by electrophoresis, immunoblotting, Enzyme-Linked Immunosorbent Assay (ELISA), protein chip, immunoprecipitation, microarray, electron microscopy, or a combination thereof. It can be. The electrophoresis may be SDS-PAGE, isoelectric electrophoresis, two-dimensional electrophoresis, or a combination thereof.

The step of detecting or measuring the expression level may include incubating the biological sample with a polynucleotide that is identical to or complementary to a polynucleotide encoding the protein variant or fragment thereof. The measuring step may be performed by Northern blotting or polymerase chain reaction (PCR). The PCR may be real-time PCR or reverse transcription PCR.

The method determines that the individual has potassium channelopathy or predicts the risk of developing it at a high level when the KCNQ4 protein variant, fragment thereof, or polynucleotide encoding the protein variant is detected in the isolated biological sample. It may include an additional step.

The step of detecting or measuring the expression level of the KCNQ4 protein variant, fragment thereof, or polynucleotide encoding the protein variant includes confirming whether the KCNQ4 protein variant is a heterozygous variant/mutation or a homozygous variant/mutation. You can.

The method is used when the KCNQ4 protein variant is a variant in which alanine and aspartic acid, which are the 271st and 272nd amino acids, are deleted, or a variant in which arginine, the 331st amino acid, is substituted with glutamine, the potassium channelopathy is caused by inactivation or low potassium channel function. It may further include a step of determining that it is due to activation, and the variant may be heterozygous or homozygous.

The method further includes the step of determining that if the KCNQ4 protein variant is a heterozygous variant in which glycine, the 319th amino acid, is substituted with aspartic acid, the potassium channelopathy is caused by hyperactivation of potassium channel function. It may be.

The method further includes the step of determining that if the KCNQ4 protein variant is a homozygous variant in which glycine, the 319th amino acid, is substituted with aspartic acid, the potassium channelopathy is caused by inactivation or hypoactivation of potassium channel function. It may be.

The method may further include the step of determining the individual as a target for treatment using a potassium channel activator when the KCNQ4 protein variant is a heterozygous variant in which arginine, the 331st amino acid, is substituted with glutamine.

The potassium channel activator is a KCNQ opener including retigabine (Ret), zinc pyrithione (ZnPy), or PIP2 (Phosphatidylinositol 4,5-phosphate) including PIP5K (Phosphatidylinositol-4-phosphate 5-kinase). bisphosphate) may be an activator.

The method may further include the step of determining the individual as a target for treatment using a potassium channel activity inhibitor when the KCNQ4 protein variant is a heterozygous variant in which glycine, the 319th amino acid, is substituted with aspartic acid.

The potassium channel activity inhibitor may include a KCNQ inhibitor including linopirdine, or a PIP2 chelating agent including polycation poly-L-lysine (PLL).

In the present invention, the novel KCNQ4 variants are a) a variant in which alanine and aspartic acid, the 271st and 272nd amino acids, are deleted (A271_D272del), b) a variant in which the 331st amino acid, arginine, is replaced with glutamine (R331Q), and c) the 319th amino acid. A variant (G319D) in which glycine was replaced with aspartic acid was discovered, and it was confirmed that by detecting this variant, dysfunction of the KCNQ4 potassium channel and non-syndromic hearing loss caused by it could be diagnosed.

In addition, in the case of the R331Q heterozygous mutation, it was confirmed that the potassium channel function was inhibited and that the channel function was recovered when treated with a potassium channel activator, while in the case of the G319D heterozygous mutation, on the contrary, the potassium channel function was overactivated and the potassium channel activity inhibitor was treated. When processed, it was confirmed that the channel function was normalized. Therefore, using this, not only can the fundamental cause of potassium channelopathy, including non-syndromic hearing loss, be identified, but also an effective treatment method can be proposed.

Another aspect is to provide a composition for screening potassium channel activity inhibitors, comprising a KCNQ4 protein and a transformant expressing a KCNQ4 protein variant in which glycine, the 319th amino acid, is substituted with aspartic acid. The same parts as described above also apply to the composition.

As used herein, the term "transformation" refers to a change in the genetic characteristics of host cells (prokaryotic and eukaryotic, animal and plant cells) by a plasmid or genome containing nucleic acids introduced from outside using genetic engineering techniques. refers to a phenomenon, and the term "transformant" refers to a cell that stably retains a gene and stably maintains gene expression even if a plasmid gene or genomic DNA introduced from outside undergoes repeated cell division. In order to perform the transformation, any method of introducing a nucleic acid into an organism, cell, tissue, organ or nucleic acid can be used, and is specifically performed by selecting an appropriate standard technique depending on the host cell as known in the art. can do.

As used herein, the term "host cell" refers to a cell into which foreign nucleic acids are introduced, specifically Escherichia coli, Bacillus subtilis , Streptomycess genus, Pseudomonas genus ( Prokaryotes such as Pseudomonassp. ), Proteus mirabilis or Staphylococcussp .; Fungi (e.g., Aspergillus sp. ), Yeasts (e.g., Pichia pastoris, Saccharomyces cerevisiae ), Schizosaccharomyce s sp. ), Neurospora crassa, etc., lower eukaryotes; cells from higher eukaryotes, including insect cells, plant cells, mammals such as CHO, HeLa, HEK293, and COS-1, etc. In addition, cultured cells (in vitro), graft cells (graft cells) and primary cell cultures (in vitro and ex vivo), commonly used in the art, and in vivo cells, and also mammalian cells, including humans.

The composition may be in the form of a stable cell line into which the KCNQ4 wild-type protein and the KCNQ4 protein variant are transfected.

The transformant may contain an expression vector containing a polynucleotide encoding the KCNQ4 wild-type protein and the KCNQ4 protein variant.

As used herein, the term “expression vector” refers to a recombinant vector that can be introduced into a suitable host cell to express a protein of interest, and refers to a genetic construct containing essential regulatory elements operably linked to express the gene insert. The term “operably linked” means that a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest are functionally linked to perform a general function. Operational linkage with a recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cutting and ligation can be easily performed using enzymes generally known in the art. there is.

A suitable expression vector of the present invention may include signal sequences for membrane targeting or secretion in addition to expression control elements such as a promoter, start codon, stop codon, polyadenylation signal, and enhancer. The initiation codon and stop codon are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and must be functional in the subject when the genetic construct is administered and must be in frame with the coding sequence. Common promoters can be constitutive or inducible and include the lac, tac, T3, and T7 promoters in prokaryotes, the simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, and human immunodeficiency virus in eukaryotes. (HIV), e.g. the long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalovirus (CMV), Epstein Barr virus (EBV), and Rouss sarcoma virus (RSV) promoters, as well as the β- Actin promoters, human heroglobin, human muscle creatine, human metallothionein-derived promoters, etc., but are not limited thereto.

Additionally, the expression vector may include a selectable marker for selecting host cells containing the vector. A selection marker is used to select cells transformed with a vector, and markers that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive, so transformed cells can be selected. Additionally, if the vector is a replicable expression vector, it may contain a replication origin, which is a specific nucleic acid sequence where replication is initiated.

Various types of vectors, such as plasmids, viruses, and cosmids, can be used as recombinant expression vectors for inserting foreign genes. The type of recombinant vector is not particularly limited as long as it functions to express the desired gene and produce the desired protein in various host cells of prokaryotic and eukaryotic cells, but specifically, it has a highly active promoter and strong expression ability while maintaining a natural state. Vectors that can produce large quantities of foreign proteins of a similar form can be used.

To express the protein of the present invention, a combination of various hosts and vectors can be used. Expression vectors suitable for eukaryotic hosts may include, but are not limited to, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. Expression vectors that can be used in bacterial hosts include, but are not limited to, pET21a, pET, pRSET, pBluescript, pGEX2T, pUC vector, col E1, pCR1, pBR322, pMB9 or their derivatives in Escherichia coli. The resulting bacterial plasmids, plasmids with a wider host range such as RP4, phage DNA, which can be exemplified by phage lambda derivatives such as λgt10, λgt11 or NM989, and others such as M13 and filamentous single-stranded DNA phages. Other DNA phages, etc. may be included. A 2°C plasmid or a derivative thereof may be used for yeast cells, and pVL941 may be used for insect cells.

The transformant co-expresses the KCNQ4 protein and the KCNQ4 protein variant, and the KCNQ4 protein and the KCNQ4 protein variant may be expressed at a ratio of 7:1 to 1:2, specifically 7:1, 6 It may be expressed at a ratio of :1, 5:1, 4:1, 3:1, 2:1, 1:1 or 1:2, and more specifically, it may be expressed at a ratio of 1:1.

The transformant may express a fusion protein containing the KCNQ4 wild-type protein and the KCNQ4 protein variant. The KCNQ4 wild-type protein and KCNQ4 protein variant may be linked directly to each other or may be linked through a linker.

The linker is not particularly limited as long as it exhibits the activities of the KCNQ4 wild-type protein and the KCNQ4 protein variant, but specifically, glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, It can be linked using amino acids such as glutamine, glutamic acid, lysine, and arginic acid. More specifically, it can be linked using multiple amino acids such as valine, leucine, aspartic acid, glycine, alanine, and proline, and one of the above amino acids. It can be used by connecting up to 20 units at a time.

In the fusion protein, the KCNQ4 protein variant may be linked to the N-terminus or C-terminus of the KCNQ4 wild-type protein, and may be specifically linked to the C-terminus.

The fusion protein is co-expressed with the KCNQ4 wild-type protein and the KCNQ4 protein variant, specifically expressed 1:1, thereby forming a KCNQ4 channel in which the wild type and the variant are assembled at a ratio of 2:2.

The KCNQ4 heterotetrameric channel assembled by co-expressing the KCNQ4 wild-type protein and a KCNQ4 protein variant in which glycine, the 319th amino acid, is substituted with aspartic acid, has a potassium channel overactivation compared to the homotetrameric channel assembled with the KCNQ4 wild-type protein, and the potassium channel It was confirmed that the activation of potassium channels was inhibited when treated with an activity inhibitor or KCNQ4 inhibitor. Therefore, potassium channel activity inhibitors can be efficiently screened using the KCNQ4 heterotetrameric channel and a transformant containing it.

Another aspect is 1) treating a transformant expressing the KCNQ4 wild-type protein and a KCNQ4 protein variant in which glycine, the 319th amino acid, is substituted with aspartic acid, with a potassium channel activity inhibitor candidate; and 2) measuring the degree of potassium channel activation of the transformant treated with the candidate material. The same parts as described above also apply to the above method.

The method may further include the step of determining that the candidate substance is a potassium channel activity inhibitor when activation of the potassium channel is inhibited by the candidate substance.

In the above method, the step of measuring the degree of potassium channel activation is electrophysiology, such as measuring the level of residual potassium ions or movement of potassium ions in the cell, or measuring the degree of change in current and/or voltage due to movement of potassium ions. This may be confirmed through scientific analysis, and specifically, it may be using patch clamps.

According to a composition for diagnosing potassium channelopathy, a kit, and a method for providing information for diagnosing potassium channelopathy using the same, the occurrence and prognosis of potassium channelopathy can be determined objectively, quickly, with high accuracy and specificity. It can be used for road detection.

Figure 1 shows the results of acoustic phenotype and sequencing analysis of a pedigree containing a novel KCNQ4 variant:
(A) Shows pure-tone audiometry results from four genealogical progenitors with different auditory phenotypes: high-frequency hearing loss (SB228 and SB155), mid-frequency hearing loss (SB356), and low-frequency hearing loss (SB62).
(B) Shows the results of Sanger sequencing chromatogram analysis by family tree:
(C) It was confirmed that novel variants are well conserved in various KCNQ4 orthologs and paralogs.
Figure 2 shows a schematic diagram of the functional domain map of KCNQ4. Specifically, the novel KCNQ4 variants identified in the present invention are p.A271_D272del located in the pore loop domain, p.G319D located in the C-terminal part of the transmembrane S6 segment, and p.R331Q located in the proximal cytoplasmic C-terminus.
Figure 3 is a diagram showing impaired channel conductance and dominant-negative effect of KCNQ4 mutant channels:
(A) Shows whole-cell K ⁺ currents recorded from HEK293T cells transiently expressing KCNQ4 WT, p.S269del, p.A271_D272del, p.G319D, p.R331Q, or GFP. In isomeric KCNQ4 mutant channels, the KCNQ4-mediated K ⁺ current, linopyrdine (30 μM)-sensitive K ⁺ current (‘subtracted’), was barely detectable.
(B) A diagram showing the current-voltage (IV) relationship of KCNQ4-mediated K ⁺ current in the concatemer of KCNQ4 WT and mutants.
(C) It was confirmed that the KCNQ opener does not activate the homologous KCNQ4 variant channel. Specifically, retigabine (Ret, 10 μM) or the combination of Ret and zinc pyrithione (Ret/ZnPy 10 μM) failed to activate the variant channel.
(D) The current density after treating the isomeric KCNQ4 mutant channel with the KCNQ opener was not significantly different from that of GFP (n = 7-9).
Figure 4 shows the dominant-negative effect of KCNQ4 variant channels.
(A) Each KCNQ4 variant (p.S269del, p.A271_D272del, p.G319D, or p.R331Q) was coexpressed with KCNQ4 wild type (WT) at the indicated molar ratio (wild type:variant), and KCNQ4-mediated K ⁺ at 40 mV. The current was measured. The dashed line below the K ⁺ current trace indicates zero current level at a holding potential of -80 mV.
(B) This is a diagram showing the current density (pA/pF) according to the molar ratio of wild type: mutant cDNA at +40mV (n=6-17), and the total amount of cDNA was the same in all experimental groups by adding an empty pRK5 vector.
(C) This is a diagram showing the degree of WT-mediated current inhibition by co-expression of KCNQ4 mutant (var.) channel. The average value of the current density at +40 mV was normalized and the rate of current inhibition was plotted against the WT/(WT + var.) cDNA molar ratio. The dashed line with square symbols indicates the expected current inhibition ratio for tetramerized channels with dominant-negative subunits.
Figure 5 shows the dominant-negative effect of KCNQ4 variant tandem concatemer channels.
(A) A schematic diagram showing the structure of the KCNQ4 mutant tandem concatemer channel.
(B) Shows whole-cell K ⁺ currents recorded from KCNQ4 channels assembled with concatemers of KCNQ4 WT and mutants. Cells transfected with GFP were used as a negative control, and linopyrdin-sensitive currents were subtracted for comparison.
(C) This is a diagram showing the current-voltage (IV) relationship of KCNQ4-mediated K ⁺ current in the concatemer of KCNQ4 WT and mutants (n = 4-21).
Figure 6 is a diagram confirming the functionality of the KCNQ4 p.A271_D272del or p.R331Q variant.
(A) This is a diagram showing the immunofluorescence observation results of HEK293T cells transfected with KCNQ4 WT, KCNQ4 p.A271_D272del, and KCNQ4 p.R331Q.
(B) This diagram shows the results of biotylation analysis on the surface of HEK293T cells transfected with KCNQ4 WT, KCNQ4 p.A271_D272del, and KCNQ4 p.R331Q (***, statistically significant).
Figure 7 is a diagram confirming whether the function of the KCNQ4 mutant channel is restored by PIP5K:
(A) The effect of PIP5K expression was compared in homomeric KCNQ4 variant channels (p.R331Q and p.G319D) and heteromeric KCNQ4 variant channels (WT-p.R331Q and WT-p.G319D) assembled with tandem concatemers. . Total K ⁺ currents were measured in the absence of PIP5K (−PIP5K) or expression of PIP5K (+PIP5K) in HEK 293T cells.
(B) Shows the IV curves of tandem concatemer channels assembled with WT-WT, WT-p.R331Q, and WT-p.G319D.
(C) Steady-state activation curves of tandem concatemer channels assembled with WT-WT, WT-p.R331Q and WT-p.G319D are shown.
(D and E) K at +40 Mv in WT, p.R331Q, p.G319D concatemer channels and WT-WT, WT-p.R331Q, and WT-p.G319D tandem concatemer channels with and without PIP5K treatment. ⁺ indicates current density and half-activation voltage (V _0.5 ). The horizontal dashed line in the graph represents the values obtained in the isogenic WT channel (WT) (n = 10-12); _** P<0.01, ***P<0.005; NS, not significant.
Figure 8 is a diagram confirming whether the function of the KCNQ4 mutant channel assembled with tandem concatemers is restored following KCNQ opener treatment.
(A) Total K ⁺ current was measured in HEK 293T cells following KCNQ opener treatment (ZnPy) in the absence of PIP5K (-PIP5K) or expression of PIP5K (+PIP5K).
(B and C) WT, p.R331Q, p.G319D concatemer channels and WT-WT, WT-p.R331Q, and WT-p.G319D tandem concatemer channels with and without PIP5K treatment and KCNQ opener (Ret 10 μM; K ⁺ current density and half-activation voltage (V _0.5 ) at +40 Mv with and without treatment (ZnPy 10 μM and ML213 3 μM) are shown. The horizontal dashed line in the graph represents the values obtained in the isomeric WT channel (WT) without PIP5K and KCNQ opener treatment (n = 10-12).
Figure 9 is a diagram confirming the effect of suppressing hyperactivation of the KCNQ4 variant channel assembled with WT-p.G319D tandem concatemer according to KCNQ inhibitor or PIP2 screening.
(A) Shows the IV curve (left) and activation curve (right) of WT-p.G319D tandem concatemer channels following linopyrdine (3-10 μM) treatment.
(B and C) IV curve (left) and activation curve (right) of WT-p.G319D tandem concatemer channels with and without PIP5K treatment and poly-L-lysine (PLL, 10 or 30 μg/ml) treatment. indicates.
Figure 10 is a diagram showing the mechanism of non-syndromic hearing loss in G319D, a novel KCNQ4 variant of the present invention.

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

Example 1: Collection of individuals (experimental group)

The procedures of all examples and experimental examples in this specification were approved by the institutional review boards of Seoul National University Hospital (IRB-H-0905-041-281) and Seoul National University Bundang Hospital (IRB-B-1007-105-402). Consent was obtained from the target patients. In the examples below, four families (SB228, SB155, SB356, and SB62) were involved, and the individuals involved underwent comprehensive phenotypic evaluation including medical history interview, physical examination, imaging, and hearing evaluation.

Example 2: Molecular genetic testing and diagnosis

Targeted panel sequencing (Otogenetics, Norcross, GA, USA) was performed using the NimbleGen Sequence Catcher (Roche NimbleGen Inc., Madison, WI, USA), initially testing for 134 known hearing loss genes in individuals lacking phenotypic markers. did. If an individual did not have a probable variant in the hearing impairment panel, exome sequencing was performed followed by bioinformatics analysis. Read results were compared to the UCSC hg19 reference genome, and non-synonymous single nucleotide polymorphisms were filtered to a depth of 40. dbSNP138 was filtered excluding flagged SNPs. Using a comprehensive filtering process, rare single nucleotide variants, indels) or splice-site variants were selected. Ethnically matched Korean Reference Genome Database (KRGDB) and ExAC (Exome Aggregation Consortium, http://exac.broadinstitute.org/ ) and gnomAD (genome aggregation database, ( http://gnomad.broadinstitute). The prevalence of mutations was confirmed using all global MAF (Minor Allele Frequency) databases, including org/ ), and then Sanger sequencing and segregation analysis were performed to identify mutations in candidate hearing loss genes.

To predict the pathogenic potential of each detected variant, in silico CADD (Combined Annotation Dependent Depletion, https://cadd.gs.washington.edu/) and REVEL (Rare Exome Variant Ensemble Learner, https:// /sites.google.com/site/revelgenomics/ ) score analysis was performed. Additionally, the evolutionary conservation of amino acid residues was determined using the GERP++ (Genomic Evolutionary Rate Profiling) score of UCSC Genome Browser. The pathogenic potential of the novel mutations in the present invention was evaluated according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines.

Example 3: Plasmid construction, cell culture and transfection

KCNQ4 WT cDNA was cloned into SgfI and KgockI sites of pEGFP-N1. Monkey embryonic kidney cell line COS-7 (Korea Culture Bank, Korea) was maintained in Dulbecco's Modified Eagle's Medium (DMEM) medium containing 10% fetal bovine serum (FBS). Before transient transfection, cells were seeded at 70-80% density in laboratory Tek II chambers. The cells were transfected with KCNQ4 wild type or mutant vector using Lipofectamine 3000 (Invitrogen, Seoul, Korea) and cultured at 37°C for 24 hours. Afterwards, the cells were stained with concanavalin A.

Example 4: Cell culture and transfection of KCNQ4 channel for electrophysiological analysis

HEK293T cells were maintained at 37°C in 5% CO ₂ conditions in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (Thermofisher). Cells were subcultured to 70-90% density and plated for transfection. One day before transfection, cells were plated at 70–90% density in 6-well culture dishes. To measure the whole-cell ionic current of the homotetrameric KCNQ4 channel, KCNQ4 WT, p.S269del, p.A271_D272del, p.G319D, and p.R331Q were cloned into the pRK5 vector, and cDNA (4.0 μg) was cloned into pEGFPN-1 ( 0.4 μg, BD Biosciences) and were transiently expressed in HEK293T cells using Lipofectamine 2000 (Invitrogen). Tandem concatemers of the KCNQ4 WT subunit or one WT and one mutant subunit were generated by fusing the subunit C-terminus to the N-terminus. Five tandem concatemers (WT-WT, WT-p.S269del, WT-p.A271_D272del, WT-p.G319D and WT-p.R331Q) fused with KCNQ4 WT were cloned into pRK5 vector and cDNA (8.0 μg) were transiently expressed in HEK293T cells with pEGFPN-1 (0.4 μg). HEK293T cells transfected with empty pRK5 vector and GFP were used as non-transfected controls (GFP). The cDNA of PIP5K (phosphatidylinositol 4-phosphate 5-kinase) cloned into the pEGFP vector was transiently expressed in HEK293T cells together with KCNQ4 cDNA. One day after transfection, cells were dissociated with 0.05% trypsin/EDTA (Thermofisher) and whole-cell patch-clamp analysis was performed.

Example 5: Whole-cell patch-clamp

KCNQ4 channel currents were measured/recorded with a conventional whole-cell patch-clamp. Patch pipettes were pulled out of borosilicate glass tubes (WPI, Sarasota, FL, USA), and pipette tips were fire-polished with a microforge (MF-83; Narishige, Japan). The final pipette tip resistance when filled with pipette solution was 1.5-3 MΩ. Whole-cell K ⁺ currents were measured and recorded with an Axopatch-1D amplifier (Axon Instrument, USA). Current was filtered at 5 KHz and collected at a sampling rate of 10 kHz. Before obtaining the ionic current, the series resistance was compensated, and the cell membrane capacitance (Cm) was measured and canceled using the circuit of the patch clamp amplifier. K ⁺ currents were generated with 2-s depolarizing voltage steps (ranging from -70 to +40 mV in 10 mV increments) followed by 1-s hyperpolarizing voltage steps at -50 mV.

To analyze the voltage-dependent gating of KCNQ4 channels, steady-state activation curves were constructed. The measured peak tail current amplitude was plotted against the pre-pulse voltage activation and the half-activation voltage (V _{0.5, act} ) was calculated by the Boltzmann function. If necessary, normalized conductivity (G/G _max ) was calculated to obtain a value of V _{0.5, act} from the IV curve. KCNQ4-mediated K ⁺ currents from total whole-cell currents were pharmacologically separated by treating cells with a KCNQ inhibitor (linopyrdine, 30 μM), and the linopyrdine-sensitive component was obtained by digital subtraction. To neutralize the negative charge of membrane PIP2, the polycationic agent poly-L-lysine (PLL, 10 or 30 μg/ml) was included in the whole-cell pipette solution. For comparison, the KCNQ4 current amplitude measured at +40 mV was divided by the measured Cm and expressed as current density (pA/pF). All patch clamp measurements/recordings were performed at room temperature (~23°C). The measured currents were analyzed using Clampex software (pCLAMP 7.0; Axon Instrument). To test the effect of KCNQ4 variants on voltage-gated channel activation, whole-cell currents in HEK293T cells transiently transfected with WT and variant proteins were measured via patch clamp recordings. Whole-cell currents for HEK293T cells transfected with empty pRK5 vector and GFP were used as negative controls.

Example 6: Chemical agents and solutions for electrophysiological analysis

The external bath solution for whole-cell voltage clamp measurements consisted of 147mM NaCl, 5mM KCl, 1.5mM _CaCl2 , _1mMMgCl2 , 10mMHEPES, and 10mM D-glucose, NMDG (N-methyl- pH was adjusted to 7.4 using D-glucamine). The internal patch pipette solution contained 130mM KCl, 10mM NaCl, 10mM EGTA, 10mM HEPES, 3mM Mg-ATP, and 0.5mM CaCl ₂ and was adjusted to pH 7.2 using KOH. The calculated free Ca ²⁺ concentration is ~10 nM. Linopirdine dihydrochloride (Tocris Bioscience), retigabine (Glentham Life Science), ML213 (Tocris Bioscience), and zinc pyrithione (Sigma-Aldrich) were dissolved in DMSO and 1,000x stock. (5-30mM) solution was prepared. The stock solution was diluted with external bath solution prior to use.

Example 7: Intracellular tracking evaluation of KCNQ4 wild type and variants

After incubation, the transfected cells were fixed in 4% paraformaldehyde for 15 minutes, and then washed with PBS three times. The fixed cells were incubated with primary antibody [ANTI-FLAG (Sigma Aldrich Corp., St. Louis, MO, USA)] at 24°C for 160 minutes, washed three times with PBS at 4°C, and incubated for a second time. The cultured cells were incubated with an antibody [F (ab ') 2-Goat anti-Mouse IgG (H + L), Invitrogen, Seoul, Korea] for 90 minutes at room temperature. The cultured cells were cultured in VECTASHIELD fixation medium (Vector Laboratories, CA, USA). ) and images were taken with a confocal microscope (Carl Zeiss, LSM710).

Example 8: Cell surface biotylation assay

^5.0 Cultivated at ℃, 5% CO ₂ . Cell surface proteins (KCNQ4 WT or mutant proteins expressed on the cell surface) were isolated using a PIERCETM cell surface protein isolation kit (Thermo Scientific) and analyzed using a Myc-tag (Abcam) based ELISA kit (Thermo Scientific).

Example 9: Statistical Analysis

Data were plotted with Origin software (version 6.1, OriginLab) and expressed as mean ± standard error of the mean (SEM), with n indicating sample number. Statistical significance was determined by paired or unpaired Student's t-test or ANOVA test, and differences at P < 0.05 were considered significant.

Experimental Example 1: Clinical phenotypic analysis of four types of KCNQ4 variants

Various audiological characteristics were observed in four families with autosomal dominant non-syndromic hearing loss 2 (DFNA2) that could be distinguished by KCNQ4 mutation type. Broadly speaking, audiological characteristics were classified into three types: high-range hearing loss (SB228 and SB155), mid-range hearing loss (SB356), and low-range hearing loss (SB62) (Figure 1A). Sensorineural hearing loss (SNHL) in SB228, SB356, and SB62 was autosomal dominant, whereas it was sporadic in SB155. In the SB228 family group, the age of onset of SNHL and the age of confirmation in the genealogical progenitor SB228-442 were in the early 20s and 33 years, respectively. Affected individuals in the SB228 family group, which includes the progenitor's siblings and father, developed high-pitched hearing in their late 20s and currently use hearing aids due to progressive hearing loss. SB155-272, the genealogical progenitor of the SB155 family group, initially showed minimal, progressive, symmetric high-frequency hearing loss in a downward sloping pattern at age 11 years. Unlike the case of SB228, in SB155 both parents had normal hearing thresholds at all frequencies. In the SB356 family, the genealogical progenitor, SB356-697 (age 33 at confirmation), presented with moderate to severe hearing loss, predominantly midrange hearing loss (symmetrical notches up to 60 dB HL at 1 kHz in both ears). Indicates the byte format. For affected individuals, there was no change in hearing thresholds over more than 1 year of follow-up. It was confirmed that the father of the genealogical progenitor had poor hearing, but it was not documented. In the SB62 family group, genealogical proband SB62-118 (58 years old at confirmation) presented with symmetrical low- and mid-range hearing loss of approximately 40 dB HL. The family shows an autosomal dominant inheritance of hearing loss. Over a 10-year follow-up period, SB62-118 showed a significant and gradual decline in hearing across all frequencies bilaterally and a decrease in speech intelligibility (speech discrimination scores). The proband's audiogram ultimately revealed a hearing threshold of 65 dB HL at all frequencies in both ears. The genealogical progenitor's mother experienced hearing loss in both ears since she was 60 years old.

Affected individuals in four families showed no signs of nystagmus or balance problems on caloric tests or video head impulse tests. No cochlear vestibular malformations were found on radiological evaluation in the affected individuals.

Experimental Example 2: Identification of new variants of KCNQ4 gene

In the four family groups identified in Example 1, four mutations that were potential causes of non-syndromic hearing loss in KCNQ4 were identified. Among these, there are three mutations, c.811_816del:p.Ala271_Asp272del (A271_D272del, SB155 family group), c.G956>A:p.G319D (G319D, SB356 family group), and c.G992>A:p.R331Q ( R331Q, SB62 family group) was confirmed to be a novel mutation (Figure 1B), and the remaining one (SB228 family group) was a previously reported in-frame deletion mutation (c.806_808del: p.Ser269del (p.S269del)). indicated. Specifically, in SB155, none of the biological parents were phenotypically affected and did not carry A271_D272del, indicating that the A271_D272del mutation arose de novo as a causal mutation in the SB155 family group. The biological parents of SB155, identified by haplotype topological analysis in the triple ES data, were phenotypically unaffected and were not carriers of the variant. The novel in-frame deletion variant A271_D272del of KCNQ4 is located in the pore loop domain (Figure 2), where most of the known KCNQ4 mutations associated with hearing loss were found in a cluster. Specifically, when six nucleic acids (GCCGAC) were deleted, alanine and aspartic acid were lost. The deletion was not present in the Korean Reference Genome Database (KRGDB, 1722 individuals), as well as in the global MAF including ExAC and gnomAD. This residue was highly conserved between KCNQ4 orthologs and paralogs (http://genome.ucsc.edu/) (Figure 1C) and gave a high GERP++ score of 5.08. The mutation has been consistently predicted to be 'disease-causing' through in silico computer simulations, including CADD. The identification of the novel occurrence of A271_D272del provides adequate evidence for PS2 in relation to hearing loss. In addition, PM1 can be applied because variant A271_D272del was present in the KCNQ4 pore-forming region, as evidenced by the well-studied functional domain without positive mutations. Therefore, variant A271_D272del has PS2_moderate, PM1, PM2 and PS3_supporting It can be classified as “likely pathogenic” according to the ACMG/AMP guidelines used to classify variants, including:

Another novel missense mutation, G319D, is located at the distal end of the transmembrane S6 segment (Figure 2) and is highly conserved among KCNQ4 orthologs and paralogs (http://genome.ucsc.edu/) (Figure 1C). , supported by a high GERP++ score of 5.27. The mutation was not found in public databases. The mutation was consistently predicted to be 'disease-causing' by in silico analysis, including CADD and REVEL. Therefore, the G319D mutation can be classified as a “variant of uncertain significance (VUS)” based on the ACMG/AMP guidelines.

Lastly, R331Q, a novel missense mutation, is the first mutation reported in the proximal cytoplasmic C-terminus (Figure 2). This residue was highly conserved between KCNQ4 orthologs and paralogs (Figure 1C), supported by the high GERP++ score of 5.44. This variant shows a very low MAF and meets the PM2 criteria. The mutation was consistently predicted as “damaging” or “probably damaging” by SIFT (Sorting Intolerant from Tolerant) and PolyPhen-2, respectively. CADD and REVEL show higher scores of 33 and 0.768 points, respectively, indicating 'disease-causing' pathogenicity. Additionally, the mutant protein displayed almost completely abolished voltage-activated potassium currents compared to the wild type (WT) protein, supporting evidence for PS3. Since the REVEL score of 0.8 is high, PP3 can also be applied. Collectively, variant R331Q was classified as “VUS” (Table 1).

The results of the analysis are summarized and listed in Table 1 below.

family group SB228-442 SB155-271 SB356-697 SB62-110 Genome location
(GRCh37/hg19) Chr1:41285116-41285118 Chr1:41285121-41285126 Chr1:41285847 Chr1:41285883 HGVS nucleotide
transition c.806_808del c.811_816del c.956G>A c.992G>A amino acid
transition p.Ser269del(p.S269del) p.Ala271_Asp272del(p.A271_D272del) p.Gly319Asp(p.G319D) p.Arg331Gln(p.R331Q) adhesion
(Zygosity) heterozygote
(heterozygote) heterozygote heterozygote heterozygote Inheritance dominant
(Dominant) Denover*
(De novo) dominant dominant In-silico predictions CADD 19.1 22.7 29.7 33 REVEL N.A. N.A. 0.938 0.919 GERP 5.08 5.08 5.27 5.44 Ethnicity MAF
- KRGDB (1722 objects) absence absence absence absence Global MAF ExAC absence absence absence absence gnomAD absence absence absence 0.000004102/1 ACMG/AMP 2018 Guidelines standard
(Criteria PM1, PM2, PM4 PS3_supporting PS2_moderate, PM1, PM2, PM4, PS3_supporting PM2, PP3, PS3_supporting PM2 PP3, PS3_supporting classification
(Classification) Likely pathogenic Likely pathogenic VUS VUS

- Abbreviation: MAF, Minor allele frequency; Het, heterozygote; VUS, variant uncertain significance; NA, not available

- Refseq transcript accession number NM_001174069.1;

- Refseq protein accession number NP_001167540

- HGVS: Human Genome Variation Society (https://www.hgvs.org/)

- Sequence Variant Nomenclature (http://varnomen.hgvs.org/)

- CADD: Combined Annotation Dependent Depletion

(https://cadd.gs.washington.edu/)

- REVEL: Rare Exome Variant Ensemble Learner

(https://sites.google.com/site/revelgenomics/)

- KRGDB: Korean Reference Genome Database

( http://coda.nih.go.kr/coda/KRGDB/index.jsp )

- ExAC: Exome Aggregation Consortium databases

- gnomAD: The Genome Aggregation Database

(https://gnomad.broadinstitute.org/)

- ACMG/AMP 2018 guidelines (http://wintervar.wglab.org/)

* The biological parents of SB155 were not phenotypically affected and were not carriers of the variant, as confirmed by haplotype topology analysis from trio exome sequencing data.

Experimental Example 3: Analysis of the impact of novel variants on KCNQ4 channel function

To determine the effect of KCNQ4 variants on voltage-gated channel activity, patch clamp recordings were used to determine whole-cell currents in HEK 293T cells transiently transfected with KCNQ4 wild-type (WT) and variant proteins. Whole cell currents for HEK 293T cells transfected with empty pRK5 vector and GFP were used as negative controls. Additionally, cells transfected with the S269del KCNQ4 variant, which is known to induce DFNA2, were used as a null function control.

KCNQ4-mediated potassium currents of WT protein displayed typical KCNQ4 channel currents with voltage-dependent slow activation and outward rectification (Figure 3A and B). Cells expressing the WT protein exhibited voltage-dependent potassium currents with a peak current density of 121.8 ± 25.4 pA/pF at +40 mV (n = 12). In contrast, the outward potassium currents produced by cells expressing KCNQ4 variant proteins (p.A271_D272del, p.R331Q and p.G319D) were barely detectable, similar to null function controls and negative controls (Figure 3B). Therefore, it can be seen that all KCNQ4 variants newly identified in the present invention completely lose their function under homologous expression conditions.

Next, the channel activity lost in the mutant protein was obtained by using the previously known KCNQ channel openers retigabine (Ret, 10 μM), zinc pyrithione (ZnPy, 10 μM), or a combination thereof (Ret/ Recovery was confirmed by ZnPy). The combination of channel openers (Ret/ZnPy) increased the KCNQ4-mediated potassium current of the WT protein by more than twofold ( Fig. 3C and D ). However, homotetrameric channels containing novel KCNQ4 variants (p.A271_D272del, p.R331Q, and p.G319D) showed almost no potassium current even with the channel opener, and the current was similar to that of the null function control and negative control. It was similar to Therefore, it can be seen that the channel function of all KCNQ4 variants newly identified in the present invention is not restored even if the KCNQ channel opener is treated under homologous expression conditions.

Experimental Example 4: Confirmation of dominant negative effects of novel KCNQ4 variants

To confirm the pathogenic mechanism of the novel KCNQ4 variant of the present invention, WT and each variant cDNA were co-expressed at various molar ratios (wild type/mutant ratio from 4:0 to 0:4) in a heteromeric system. Potassium currents were measured in HEK 293T cells.

As a result, the generated KCNQ4-mediated potassium current was found to be inversely proportional to the concentration of the mutant cDNA (Figure 4A and B), without a clear dominant-negative effect of the mutant protein on the WT protein: KCNQ4WT:Vector (2:2). Peak current density: 131.7 ± 21.1 pA/pF at +40 mV, n=11; Peak current density of WT:p.A271_D272del (2:2): 69.8 ± 19.9 pA/pF at +40 mV, n=7; WT: Peak current density of p.G319D (2:2): 152.7 ± 45.7 pA/pF at +40 mV, n=11; WT: Peak current density of p.R331Q (2:2): 55.1 ± 17.8 pA/pF at + 40 mV, n=6.

Excluding the null function control (p. S269del pore variant), the novel KCNQ4 variant channels deviated from the predicted channel function inhibition ratios for WT and co-assembled tetramers containing the dominant-negative subunit. For example, WT:p.G319D-mediated currents deviated significantly from predictions, showing larger currents than WT channels at 3:1 or 2:2 cDNA ratios. Additionally, firm conclusions could not be made due to the variance and outliers of current amplitude within the same group (Figure 4C). Summarizing the above results, it can be seen that channels co-expressed with the novel KCNQ4 variant and WT do not necessarily show a general dominant-negative effect.

Experimental Example 5: Production of KCNQ4 variant tandem concatemer and confirmation of its dominant-negative effect

In order to more clearly confirm the dominant-negative effect of the heteromeric channel containing KCNQ4 WT and variants confirmed in Example 4 above, the variants and WT were included in a ratio of 2:2 (i.e., with uniform heterotetramerization) A tandem concatemer was constructed in which KCNQ4 WT and the mutant were linked (Figure 5A). The concatemer was constructed so that the WT was located at the N-terminus and the mutant was located at the C-terminus.

As a result of confirming the dominant-negative effect using the KCNQ4 concatemer channel produced above, the WT-p.A271_D272del concatemer channel was generated by the null functional control (WT-p.S269del concatemer) and the negative GFP control. Similar to what was seen, little potassium current was detectable, indicating a non-conducting KCNQ4 variant channel (Figure 5B). The KCNQ4-mediated potassium current of the WT-p.R331Q concatemer was slightly larger than that of the isoform p.R331Q, but statistical significance was not achieved and the amplitude was much lower than the value of the WT-WT concatemer (Figure 5B and C ). On the other hand, some cells expressing the WT-p.R331Q concatemer showed larger currents than cells expressing the WT-p.S269del concatemer and negative GFP, which may be explained by inter-individual cell-to-cell variability in membrane phospholipid levels. You can.

Taking the above results together, p.A271_D272del, a novel pore region variant, exerts a dominant negative effect on WT channels, while p.R331Q is a variant that can restore part of the function when assembled with WT protein under limited conditions. Able to know. Cell surface expression analysis showed that the two KCNQ4 variant proteins successfully reached the plasma membrane and their expression levels were similar to the WT protein, except for defective plasma membrane trafficking due to the pathogenicity of these variants (Figure 6).

Experimental Example 6: Potassium channel activation analysis of WT-p.G319D concatemer channel

As confirmed in Example 4 above, when WT and p.R331D mutant cDNA were co-transfected, the potassium current of the heteromeric p.G319D channel was higher than that of the homomeric WT channel. Therefore, to confirm the dominant-negative effect of p.R331D, a novel KCNQ4 variant, the function of the WT-p.G319D concatemer channel was analyzed.

As a result, the WT-p.G319D concatemer had a lower mean value of KCNQ4-mediated current (174.9 ± 51.5 pA/pF at +40 mV, P < 0.05) compared to the WT-WT concatemer (95.2 ± 10.4 pA/pF). There was a statistically significant difference. (Figure 5C).

Next, we analyzed the channel functionality for WT-WT, WT-p.G331Q and WT-p.G319D concatemer channels and found that the total potassium current of WT-p.G319D concatemer (198.7 ± 15.0 at +40 mV) pA/pF) was approximately twice that of WT-WT concatemer (110.2 ± 7.8 pA/pF) (Figure 7A and D). The WT-p.G319D concatemer showed a significant shift in the current-voltage (IV) relationship and a voltage dependence of activation on negative potentials (Figure 7B). The calculated half-activation voltage (V _0.5 ) of the WT-p.G319D concatemer was -38.2 ± 0.6 mV, and the V _0.5 of the WT-WT concatemer was -21.1 ± 1.0 Mv (Figure 7C and E). Based on the above results, it can be seen that p.G319D, unlike other novel variants, does not exhibit a dominant-negative inhibitory effect on WT channels, but rather is a gain-of-function mutation (gain-of-function) when heterotetramerized with the KCNQ4 wild-type subunit. It is judged to be an active DFNA2 variant with a certain function.

Experimental Example 7: Confirmation of functional recovery according to PIP5 and KCNQ opener processing of KCNQ4 mutant channel

To determine whether the dysfunction of KCNQ4 mutant channels could be restored, WT-WT, WT-p.G331Q, and WT-p.G319D concatemer channels were treated with PIP5K and KCNQ opener, which are known to activate KCNQ channels. Afterwards, it was checked whether the channel function was restored.

As a result, when the PIP2 concentration was increased by expressing PIP5K, the WT and WT-WT channel currents were improved by more than 2-fold, and the V _0.5 value shifted toward the negative charge by about -10 mV (FIG. 7). In conjunction with PIP5K expression, treatment with the KCNQ channel opener (10 μM of Ret, 10 μM of ZnPy, and 3 μM of ML213) further enhanced the channel current, but no stronger enhancement effect was observed with the combined treatment (Figure 8). These results indicate that the KCNQ4 WT concatemer channel and KCNQ4 WT-WT concatemer channel respond to the KCNQ opener while maintaining sensitivity to PIP2.

In contrast, the two pore domain variants (p.S269del and p.A271_D272del) in the concatemer form (WT-p.S269del and WT-p.A271_D272del) were activated when treated with PIP5K, KCNQ channel opener, or a combination of the two. Even then, channel function was not recovered (Figures 7 and 8).

Next, for the p.R331Q mutant, the impaired conductance of the homomeric p.R331Q mutant channel was not restored upon treatment with PIP5K, the KCNQ channel opener, or a combination of both, but in contrast to the heteromeric WT-p.R331Q concatenate. When the mer channel was treated with PIP5K, KCNQ channel opener, or a combination of both, channel function was restored, as evidenced by a shift in the V _0.5 value toward the negative charge (Figures 7 and 8). Specifically, PIP5K expression alone restored the conductance of the WT-p.R331Q concatemer, showing results similar to the current of normal isomeric WT or WT-WT concatemer channels ( Fig. 7B to E ). Additionally, even in the absence of PIP5K expression, three KNCQ openers (Ret 10 μM, ZnPy 10 μM, and ML213 3 μM) significantly increased the conductance of the WT-p.R331Q concatemer up to 70% of the normal WT current ( Fig. 8A to B ). . When PIP5K was expressed, activation of the WT-p.R331Q concatemer by the KCNQ opener was prominent, and currents reached normal WT or WT-WT current levels measured in the absence of PIP5K (Figure 8B-C). Taking the above results together, it can be seen that the function of the pathogenic heteromeric WT-p.R331Q channel can be restored by increased PIP2 concentration, KCNQ opener drug, or both.

Next, in the case of the p.R319D mutation, the channel function of the non-conducting isoform p.G319D mutant channel was not restored by PIP5K or KCNQ opener or a combination of the two. On the other hand, hyperactivated WT-p.G319D concatemer channels responded to the PIP5K or KCNQ opener or both, and, like the WT-WT and WT-p.R331Q concatemers, WT-p by the PIP5K or KCNQ opener. The increase in current of the .G319D concatemer was proportional to the degree of shift of V _0.5 toward the negative charge, indicating that PIP2 activation was mediated by promotion of voltage-dependent channel gating (Figures 7 and 8).

Normalizing hyperactivated WT-pG319D channels means reducing channel activity to normal WT levels. In order to confirm whether normalization as described above is possible, the WT-p.G319D concatemer channel was treated with a KCNQ inhibitor and a PIP2 chelating agent to confirm the current reduction effect. When treated with linopyrdine, a previously known KCNQ inhibitor, it was confirmed that the V _0.5 value shifted toward the positive potential. It was found that treatment with the KCNQ inhibitor can reduce hyperactivated WT-p.G319D-mediated current. (Figure 9A). Interference of PIP2 activation in WT-G319D channels by the intracellularly applied PIP2 chelating agent PLL (polycation poly-L-lysine, 10 or 30 μg/ml), with or without PIP5K expression conditions, resulted in a positive V value of _0.5 . By moving to , it can be seen that the current is reduced (FIGS. 9B-C). However, when only PIP2 was deleted, the increased channel activity was not completely restored to WT levels. PLL-treated cells showed 40% higher potassium currents than WT concatemer channels, and V _0.5 was located at -10 mV negative side compared to WT-WT. As a result, KCNQ inhibitors were more effective in reducing channel current and PIP2 chelators were more effective in normalizing the voltage dependence of channel activation. Therefore, based on the above results, it can be seen that the hyperactivated channel conductance of the heteromeric WT-p.G319D can be down-regulated through a KCNQ inhibitor (linopyrdine), inhibition of PIP2 activation, or a combination thereof.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

<110> SEOUL NATIONAL UNIVERSITY HOSPITAL <120> Novel KCNQ4 protein variant and use thereof <130> PN137403KR <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 695 <212> PRT <213> Artificial Sequence <220> <223> KCNQ4_AA <400> 1 Met Ala Glu Ala Pro Pro Arg Arg Leu Gly Leu Gly Pro Pro Pro Gly 1 5 10 15 Asp Ala Pro Arg Ala Glu Leu Val Ala Leu Thr Ala Val Gln Ser Glu 20 25 30 Gln Gly Glu Ala Gly Gly Gly Gly Ser Pro Arg Arg Leu Gly Leu Leu 35 40 45 Gly Ser Pro Leu Pro Pro Gly Ala Pro Leu Pro Gly Pro Gly Ser Gly 50 55 60 Ser Gly Ser Ala Cys Gly Gln Arg Ser Ser Ala Ala His Lys Arg Tyr 65 70 75 80 Arg Arg Leu Gln Asn Trp Val Tyr Asn Val Leu Glu Arg Pro Arg Gly 85 90 95 Trp Ala Phe Val Tyr His Val Phe Ile Phe Leu Leu Val Phe Ser Cys 100 105 110 Leu Val Leu Ser Val Leu Ser Thr Ile Gln Glu His Gln Glu Leu Ala 115 120 125 Asn Glu Cys Leu Leu Ile Leu Glu Phe Val Met Ile Val Val Phe Gly 130 135 140 Leu Glu Tyr Ile Val Arg Val Trp Ser Ala Gly Cys Cys Cys Arg Tyr 145 150 155 160 Arg Gly Trp Gln Gly Arg Phe Arg Phe Ala Arg Lys Pro Phe Cys Val 165 170 175 Ile Asp Phe Ile Val Phe Val Ala Ser Val Ala Val Ile Ala Ala Gly 180 185 190 Thr Gln Gly Asn Ile Phe Ala Thr Ser Ala Leu Arg Ser Met Arg Phe 195 200 205 Leu Gln Ile Leu Arg Met Val Arg Met Asp Arg Arg Gly Gly Thr Trp 210 215 220 Lys Leu Leu Gly Ser Val Val Tyr Ala His Ser Lys Glu Leu Ile Thr 225 230 235 240 Ala Trp Tyr Ile Gly Phe Leu Val Leu Ile Phe Ala Ser Phe Leu Val 245 250 255 Tyr Leu Ala Glu Lys Asp Ala Asn Ser Asp Phe Ser Ser Tyr Ala Asp 260 265 270 Ser Leu Trp Trp Gly Thr Ile Thr Leu Thr Thr Ile Gly Tyr Gly Asp 275 280 285 Lys Thr Pro His Thr Trp Leu Gly Arg Val Leu Ala Ala Gly Phe Ala 290 295 300 Leu Leu Gly Ile Ser Phe Phe Ala Leu Pro Ala Gly Ile Leu Gly Ser 305 310 315 320 Gly Phe Ala Leu Lys Val Gln Glu Gln His Arg Gln Lys His Phe Glu 325 330 335 Lys Arg Arg Met Pro Ala Ala Asn Leu Ile Gln Ala Ala Trp Arg Leu 340 345 350 Tyr Ser Thr Asp Met Ser Arg Ala Tyr Leu Thr Ala Thr Trp Tyr Tyr 355 360 365 Tyr Asp Ser Ile Leu Pro Ser Phe Arg Glu Leu Ala Leu Leu Phe Glu 370 375 380 His Val Gln Arg Ala Arg Asn Gly Gly Leu Arg Pro Leu Glu Val Arg 385 390 395 400 Arg Ala Pro Val Pro Asp Gly Ala Pro Ser Arg Tyr Pro Pro Val Ala 405 410 415 Thr Cys His Arg Pro Gly Ser Thr Ser Phe Cys Pro Gly Glu Ser Ser 420 425 430 Arg Met Gly Ile Lys Asp Arg Ile Arg Met Gly Ser Ser Gln Arg Arg 435 440 445 Thr Gly Pro Ser Lys Gln His Leu Ala Pro Pro Thr Met Pro Thr Ser 450 455 460 Pro Ser Ser Glu Gln Val Gly Glu Ala Thr Ser Pro Thr Lys Val Gln 465 470 475 480 Lys Ser Trp Ser Phe Asn Asp Arg Thr Arg Phe Arg Ala Ser Leu Arg 485 490 495 Leu Lys Pro Arg Thr Ser Ala Glu Asp Ala Pro Ser Glu Glu Val Ala 500 505 510 Glu Glu Lys Ser Tyr Gln Cys Glu Leu Thr Val Asp Asp Ile Met Pro 515 520 525 Ala Val Lys Thr Val Ile Arg Ser Ile Arg Ile Leu Lys Phe Leu Val 530 535 540 Ala Lys Arg Lys Phe Lys Glu Thr Leu Arg Pro Tyr Asp Val Lys Asp 545 550 555 560 Val Ile Glu Gln Tyr Ser Ala Gly His Leu Asp Met Leu Gly Arg Ile 565 570 575 Lys Ser Leu Gln Thr Arg Val Asp Gln Ile Val Gly Arg Gly Pro Gly 580 585 590 Asp Arg Lys Ala Arg Glu Lys Gly Asp Lys Gly Pro Ser Asp Ala Glu 595 600 605 Val Val Asp Glu Ile Ser Met Met Gly Arg Val Val Lys Val Glu Lys 610 615 620 Gln Val Gln Ser Ile Glu His Lys Leu Asp Leu Leu Leu Gly Phe Tyr 625 630 635 640 Ser Arg Cys Leu Arg Ser Gly Thr Ser Ala Ser Leu Gly Ala Val Gln 645 650 655 Val Pro Leu Phe Asp Pro Asp Ile Thr Ser Asp Tyr His Ser Pro Val 660 665 670 Asp His Glu Asp Ile Ser Val Ser Ala Gln Thr Leu Ser Ile Ser Arg 675 680 685 Ser Val Ser Thr Asn Met Asp 690 695 <210> 2 <211> 2088 <212> DNA <213> Artificial Sequence <220> <223> KCNQ4_NA <400> 2 atggccgagg cccccccgcg ccgcctcggc ctgggtcccc cgcccgggga cgccccccgc 60 gcggagctag tggcgctcac ggccgtgcag agcgaacagg gcgaggcggg cgggggcggc 120 tccccgcgcc gcctcggcct cctgggcagc cccctgccgc cgggcgcgcc cctccctggg 180 ccgggctccg gctcgggctc cgcctgcggc cagcgctcct cggccgcgca caagcgctac 240 cgccgcctgc agaactgggt ctacaacgtg ctggagcggc cccgcggctg ggccttcgtc 300 taccacgtct tcatattttt gctggtcttc agctgcctgg tgctgtctgt gctgtccact 360 atccaggagc accaggaact tgccaacgag tgtctcctca tcttggaatt cgtgatgatc 420 gtggttttcg gcttggagta catcgtccgg gtctggtccg ccggatgctg ctgccgctac 480 cgaggatggc agggtcgctt ccgctttgcc agaaagccct tctgtgtcat cgacttcatc 540 gtgttcgtgg cctcggtggc cgtcatcgcc gcgggtaccc a gggcaacat cttcgccacg 600 tccgcgctgc gcagcatgcg cttcctgcag atcctgcgca tggtgcgcat ggaccgccgc 660 ggcggcacct ggaagctgct gggctcagtg gtctacgcgc atagcaagga gctgatcacc 720 gcctggtaca tcgggttcct ggtgctcatc ttcgcctcct tcctggtcta cctggctgag 780 aaggacgcca actccgactt ctcctcctac gccgactcgc tctggtgggg gacgattaca 840 ttgacaacca tc ggctatgg tgacaagaca ccgcacacat ggctgggcag ggtcctggct 900 gctggcttcg ccttactggg catctctttc tttgccctgc ctgccggcat cctaggctcc 960 ggctttgccc tgaaggtcca ggagcagcac cggcagaagc acttcgagaa gcggaggatg 1020 ccgg cagcca acctcatcca ggctgcctgg cgcctgtact ccaccgatat gagccgggcc 1080 tacctgacag ccacctggta ctactatgac agtatcctcc catccttcag agagctggcc 1140 ctcttgtttg agcacgtgca acgggcccgc aatgggggcc tacggcccct ggaggtgcgg 1200 cgggcgccgg tacccgacgg agcaccctcc cgttaccccgc ccgttgccac ctgccaccgg 1260 ccgggca gca cctccttctg ccctggggaa agcagccgga tgggcatcaa agaccgcatc 1320 cgcatgggca gctcccagcg gcggacgggt ccttccaagc agcatctggc acctccaaca 1380 atgcccacct ccccaagcag cgagcaggtg ggtgaggcca ccagccccac caaggtgcaa 14 40 aagagctgga gcttcaatga ccgcacccgc ttccgggcat ctctgagact caaaccccgc 1500 acctctgctg aggatgcccc ctcagaggaa gtagcagagg agaagagcta ccagtgtgag 1560 ctcacggtgg acgacatcat gcctgctgtg aagacagtca tccgctccat caggattctc 1620 aagttcctgg tggccaaaag gaaattcaag gagacactgc gaccgtacga cgtgaaggac 1680 gtcattgagc agtactcagc aggccacctg gacatgctgg gccggatcaa gagcctgcaa 1740 actcgggtgg accaaattgt gggtcggggg cccggggaca ggaaggcccg ggagaagggc 1800 gacaaggggc cctccgacgc ggaggtggtg gatgaaatca gcatgatggg acgcgtggtc 1860 aaggtggaga agcaggtg ca gtccatcgag cacaagctgg acctgctgtt gggcttctat 1920 tcgcgctgcc tgcgctctgg cacctcggcc agcctgggcg ccgtgcaagt gccgctgttc 1980 gaccccgaca tcacctccga ctaccacagc cctgtggacc acgaggacat ctccgtctcc 2040 gcacagacgc tcagcatctc ccgctcggtc agcaccaaca tggactga 2088 <210> 3 <211> 693 <212> PRT <213> Artificial S equence <220> <223> KCNQ4_A271_D272del_AA <400> 3 Met Ala Glu Ala Pro Pro Arg Arg Leu Gly Leu Gly Pro Pro Pro Gly 1 5 10 15 Asp Ala Pro Arg Ala Glu Leu Val Ala Leu Thr Ala Val Gln Ser Glu 20 25 30 Gln Gly Glu Ala Gly Gly Gly Gly Ser Pro Arg Arg Leu Gly Leu Leu 35 40 45 Gly Ser Pro Leu Pro Pro Gly Ala Pro Leu Pro Gly Pro Gly Ser Gly 50 55 60 Ser Gly Ser Ala Cys Gly Gln Arg Ser Ser Ala Ala His Lys Arg Tyr 65 70 75 80 Arg Arg Leu Gln Asn Trp Val Tyr Asn Val Leu Glu Arg Pro Arg Gly 85 90 95 Trp Ala Phe Val Tyr His Val Phe Ile Phe Leu Leu Val Phe Ser Cys 100 105 110 Leu Val Leu Ser Val Leu Ser Thr Ile Gln Glu His Gln Glu Leu Ala 115 120 125 Asn Glu Cys Leu Leu Ile Leu Glu Phe Val Met Ile Val Val Phe Gly 130 135 140 Leu Glu Tyr Ile Val Arg Val Trp Ser Ala Gly Cys Cys Cys Arg Tyr 145 150 155 160 Arg Gly Trp Gln Gly Arg Phe Arg Phe Ala Arg Lys Pro Phe Cys Val 165 170 175 Ile Asp Phe Ile Val Phe Val Ala Ser Val Ala Val Ile Ala Ala Gly 180 185 190 Thr Gln Gly Asn Ile Phe Ala Thr Ser Ala Leu Arg Ser Met Arg Phe 195 200 205 Leu Gln Ile Leu Arg Met Val Arg Met Asp Arg Gly Gly Thr Trp 210 215 220 Lys Leu Leu Gly Ser Val Val Tyr Ala His Ser Lys Glu Leu Ile Thr 225 230 235 240 Ala Trp Tyr Ile Gly Phe Leu Val Leu Ile Phe Ala Ser Phe Leu Val 245 250 255 Tyr Leu Ala Glu Lys Asp Ala Asn Ser Asp Phe Ser Ser Tyr Ser Leu 260 265 270 Trp Trp Gly Thr Ile Thr Leu Thr Thr Ile Gly Tyr Gly Asp Lys Thr 275 280 285 Pro His Thr Trp Leu Gly Arg Val Leu Ala Ala Gly Phe Ala Leu Leu 290 295 300 Gly Ile Ser Phe Phe Ala Leu Pro Ala Gly Ile Leu Gly Ser Gly Phe 305 310 315 320 Ala Leu Lys Val Gln Glu Gln His Arg Gln Lys His Phe Glu Lys Arg 325 330 335 Arg Met Pro Ala Ala Asn Leu Ile Gln Ala Ala Trp Arg Leu Tyr Ser 340 345 350 Thr Asp Met Ser Arg Ala Tyr Leu Thr Ala Thr Trp Tyr Tyr Tyr Asp 355 360 365 Ser Ile Leu Pro Ser Phe Arg Glu Leu Ala Leu Leu Phe Glu His Val 370 375 380 Gln Arg Ala Arg Asn Gly Gly Leu Arg Pro Leu Glu Val Arg Arg Ala 385 390 395 400 Pro Val Pro Asp Gly Ala Pro Ser Arg Tyr Pro Pro Val Ala Thr Cys 405 410 415 His Arg Pro Gly Ser Thr Ser Phe Cys Pro Gly Glu Ser Ser Arg Met 420 425 430 Gly Ile Lys Asp Arg Ile Arg Met Gly Ser Ser Gln Arg Arg Thr Gly 435 440 445 Pro Ser Lys Gln His Leu Ala Pro Pro Thr Met Pro Thr Ser Pro Ser 450 455 460 Ser Glu Gln Val Gly Glu Ala Thr Ser Pro Thr Lys Val Gln Lys Ser 465 470 475 480 Trp Ser Phe Asn Asp Arg Thr Arg Phe Arg Ala Ser Leu Arg Leu Lys 485 490 495 Pro Arg Thr Ser Ala Glu Asp Ala Pro Ser Glu Glu Val Ala Glu Glu 500 505 510 Lys Ser Tyr Gln Cys Glu Leu Thr Val Asp Asp Ile Met Pro Ala Val 515 520 525 Lys Thr Val Ile Arg Ser Ile Arg Ile Leu Lys Phe Leu Val Ala Lys 530 535 540 Arg Lys Phe Lys Glu Thr Leu Arg Pro Tyr Asp Val Lys Asp Val Ile 545 550 555 560 Glu Gln Tyr Ser Ala Gly His Leu Asp Met Leu Gly Arg Ile Lys Ser 565 570 575 Leu Gln Thr Arg Val Asp Gln Ile Val Gly Arg Gly Pro Gly Asp Arg 580 585 590 Lys Ala Arg Glu Lys Gly Asp Lys Gly Pro Ser Asp Ala Glu Val Val 595 600 605 Asp Glu Ile Ser Met Met Gly Arg Val Val Lys Val Glu Lys Gln Val 610 615 620 Gln Ser Ile Glu His Lys Leu Asp Leu Leu Leu Gly Phe Tyr Ser Arg 625 630 635 640 Cys Leu Arg Ser Gly Thr Ser Ala Ser Leu Gly Ala Val Gln Val Pro 645 650 655 Leu Phe Asp Pro Asp Ile Thr Ser Asp Tyr His Ser Pro Val Asp His 660 665 670 Glu Asp Ile Ser Val Ser Ala Gln Thr Leu Ser Ile Ser Arg Ser Val 675 680 685 Ser Thr Asn Met Asp 690 <210> 4 <211> 695 <212> PRT <213> Artificial Sequence <220> <223> KCNQ4_R331Q_AA < 400> 4 Met Ala Glu Ala Pro Pro Arg Arg Leu Gly Leu Gly Pro Pro Pro Gly 1 5 10 15 Asp Ala Pro Arg Ala Glu Leu Val Ala Leu Thr Ala Val Gln Ser Glu 20 25 30 Gln Gly Glu Ala Gly Gly Gly Gly Ser Pro Arg Arg Leu Gly Leu Leu 35 40 45 Gly Ser Pro Leu Pro Pro Gly Ala Pro Leu Pro Gly Pro Gly Ser Gly 50 55 60 Ser Gly Ser Ala Cys Gly Gln Arg Ser Ser Ala Ala His Lys Arg Tyr 65 70 75 80 Arg Arg Leu Gln Asn Trp Val Tyr Asn Val Leu Glu Arg Pro Arg Gly 85 90 95 Trp Ala Phe Val Tyr His Val Phe Ile Phe Leu Leu Val Phe Ser Cys 100 105 110 Leu Val Leu Ser Val Leu Ser Thr Ile Gln Glu His Gln Glu Leu Ala 115 120 125 Asn Glu Cys Leu Leu Ile Leu Glu Phe Val Met Ile Val Val Phe Gly 130 135 140 Leu Glu Tyr Ile Val Arg Val Trp Ser Ala Gly Cys Cys Cys Arg Tyr 145 150 155 160 Arg Gly Trp Gln Gly Arg Phe Arg Phe Ala Arg Lys Pro Phe Cys Val 165 170 175 Ile Asp Phe Ile Val Phe Val Ala Ser Val Ala Val Ile Ala Ala Gly 180 185 190 Thr Gln Gly Asn Ile Phe Ala Thr Ser Ala Leu Arg Ser Met Arg Phe 195 200 205 Leu Gln Ile Leu Arg Met Val Arg Met Asp Arg Arg Gly Gly Thr Trp 210 215 220 Lys Leu Leu Gly Ser Val Val Tyr Ala His Ser Lys Glu Leu Ile Thr 225 230 235 240 Ala Trp Tyr Ile Gly Phe Leu Val Leu Ile Phe Ala Ser Phe Leu Val 245 250 255 Tyr Leu Ala Glu Lys Asp Ala Asn Ser Asp Phe Ser Ser Tyr Ala Asp 260 265 270 Ser Leu Trp Trp Gly Thr Ile Thr Leu Thr Thr Ile Gly Tyr Gly Asp 275 280 285 Lys Thr Pro His Thr Trp Leu Gly Arg Val Leu Ala Ala Gly Phe Ala 290 295 300 Leu Leu Gly Ile Ser Phe Phe Ala Leu Pro Ala Gly Ile Leu Gly Ser 305 310 315 320 Gly Phe Ala Leu Lys Val Gln Glu Gln His Gln Gln Lys His Phe Glu 325 330 335 Lys Arg Arg Met Pro Ala Ala Asn Leu Ile Gln Ala Ala Trp Arg Leu 340 345 350 Tyr Ser Thr Asp Met Ser Arg Ala Tyr Leu Thr Ala Thr Trp Tyr Tyr 355 360 365 Tyr Asp Ser Ile Leu Pro Ser Phe Arg Glu Leu Ala Leu Leu Phe Glu 370 375 380 His Val Gln Arg Ala Arg Asn Gly Gly Leu Arg Pro Leu Glu Val Arg 385 390 395 400 Arg Ala Pro Val Pro Asp Gly Ala Pro Ser Arg Tyr Pro Pro Val Ala 405 410 415 Thr Cys His Arg Pro Gly Ser Thr Ser Phe Cys Pro Gly Glu Ser Ser 420 425 430 Arg Met Gly Ile Lys Asp Arg Ile Arg Met Gly Ser Ser Gln Arg Arg 435 440 445 Thr Gly Pro Ser Lys Gln His Leu Ala Pro Pro Thr Met Pro Thr Ser 450 455 460 Pro Ser Ser Glu Gln Val Gly Glu Ala Thr Ser Pro Thr Lys Val Gln 465 470 475 480 Lys Ser Trp Ser Phe Asn Asp Arg Thr Arg Phe Arg Ala Ser Leu Arg 485 490 495 Leu Lys Pro Arg Thr Ser Ala Glu Asp Ala Pro Ser Glu Glu Val Ala 500 505 510 Glu Glu Lys Ser Tyr Gln Cys Glu Leu Thr Val Asp Asp Ile Met Pro 515 520 525 Ala Val Lys Thr Val Ile Arg Ser Ile Arg Ile Leu Lys Phe Leu Val 530 535 540 Ala Lys Arg Lys Phe Lys Glu Thr Leu Arg Pro Tyr Asp Val Lys Asp 545 550 555 560 Val Ile Glu Gln Tyr Ser Ala Gly His Leu Asp Met Leu Gly Arg Ile 565 570 575 Lys Ser Leu Gln Thr Arg Val Asp Gln Ile Val Gly Arg Gly Pro Gly 580 585 590 Asp Arg Lys Ala Arg Glu Lys Gly Asp Lys Gly Pro Ser Asp Ala Glu 595 600 605 Val Val Asp Glu Ile Ser Met Met Gly Arg Val Val Lys Val Glu Lys 610 615 620 Gln Val Gln Ser Ile Glu His Lys Leu Asp Leu Leu Leu Gly Phe Tyr 625 630 635 640 Ser Arg Cys Leu Arg Ser Gly Thr Ser Ala Ser Leu Gly Ala Val Gln 645 650 655 Val Pro Leu Phe Asp Pro Asp Ile Thr Ser Asp Tyr His Ser Pro Val 660 665 670 Asp His Glu Asp Ile Ser Val Ser Ala Gln Thr Leu Ser Ile Ser Arg 675 680 685 Ser Val Ser Thr Asn Met Asp 690 695 <210> 5 <211> 695 <212> PRT <213> Artificial Sequence <220> <223> KCNQ4_G319D_AA <400> 5 Met Ala Glu Ala Pro Pro Arg Arg Leu Gly Leu Gly Pro Pro Pro Gly 1 5 10 15 Asp Ala Pro Arg Ala Glu Leu Val Ala Leu Thr Ala Val Gln Ser Glu 20 25 30 Gln Gly Glu Ala Gly Gly Gly Gly Ser Pro Arg Arg Leu Gly Leu Leu 35 40 45 Gly Ser Pro Leu Pro Pro Gly Ala Pro Leu Pro Gly Pro Gly Ser Gly 50 55 60 Ser Gly Ser Ala Cys Gly Gln Arg Ser Ser Ala Ala His Lys Arg Tyr 65 70 75 80 Arg Arg Leu Gln Asn Trp Val Tyr Asn Val Leu Glu Arg Pro Arg Gly 85 90 95 Trp Ala Phe Val Tyr His Val Phe Ile Phe Leu Leu Val Phe Ser Cys 100 105 110 Leu Val Leu Ser Val Leu Ser Thr Ile Gln Glu His Gln Glu Leu Ala 115 120 125 Asn Glu Cys Leu Leu Ile Leu Glu Phe Val Met Ile Val Val Phe Gly 130 135 140 Leu Glu Tyr Ile Val Arg Val Trp Ser Ala Gly Cys Cys Cys Arg Tyr 145 150 155 160 Arg Gly Trp Gln Gly Arg Phe Arg Phe Ala Arg Lys Pro Phe Cys Val 165 170 175 Ile Asp Phe Ile Val Phe Val Ala Ser Val Ala Val Ile Ala Ala Gly 180 185 190 Thr Gln Gly Asn Ile Phe Ala Thr Ser Ala Leu Arg Ser Met Arg Phe 195 200 205 Leu Gln Ile Leu Arg Met Val Arg Met Asp Arg Arg Gly Gly Thr Trp 210 215 220 Lys Leu Leu Gly Ser Val Val Tyr Ala His Ser Lys Glu Leu Ile Thr 225 230 235 240 Ala Trp Tyr Ile Gly Phe Leu Val Leu Ile Phe Ala Ser Phe Leu Val 245 250 255 Tyr Leu Ala Glu Lys Asp Ala Asn Ser Asp Phe Ser Ser Tyr Ala Asp 260 265 270 Ser Leu Trp Trp Gly Thr Ile Thr Leu Thr Ile Gly Tyr Gly Asp 275 280 285 Lys Thr Pro His Thr Trp Leu Gly Arg Val Leu Ala Ala Gly Phe Ala 290 295 300 Leu Leu Gly Ile Ser Phe Phe Ala Leu Pro Ala Gly Ile Leu Asp Ser 305 310 315 320 Gly Phe Ala Leu Lys Val Gln Glu Gln His Arg Gln Lys His Phe Glu 325 330 335 Lys Arg Arg Met Pro Ala Ala Asn Leu Ile Gln Ala Ala Trp Arg Leu 340 345 350 Tyr Ser Thr Asp Met Ser Arg Ala Tyr Leu Thr Ala Thr Trp Tyr Tyr 355 360 365 Tyr Asp Ser Ile Leu Pro Ser Phe Arg Glu Leu Ala Leu Leu Phe Glu 370 375 380 His Val Gln Arg Ala Arg Asn Gly Gly Leu Arg Pro Leu Glu Val Arg 385 390 395 400 Arg Ala Pro Val Pro Asp Gly Ala Pro Ser Arg Tyr Pro Pro Val Ala 405 410 415 Thr Cys His Arg Pro Gly Ser Thr Ser Phe Cys Pro Gly Glu Ser Ser 420 425 430 Arg Met Gly Ile Lys Asp Arg Ile Arg Met Gly Ser Ser Gln Arg Arg 435 440 445 Thr Gly Pro Ser Lys Gln His Leu Ala Pro Pro Thr Met Pro Thr Ser 450 455 460 Pro Ser Ser Glu Gln Val Gly Glu Ala Thr Ser Pro Thr Lys Val Gln 465 470 475 480 Lys Ser Trp Ser Phe Asn Asp Arg Thr Arg Phe Arg Ala Ser Leu Arg 485 490 495 Leu Lys Pro Arg Thr Ser Ala Glu Asp Ala Pro Ser Glu Glu Val Ala 500 505 510 Glu Glu Lys Ser Tyr Gln Cys Glu Leu Thr Val Asp Asp Ile Met Pro 515 520 525 Ala Val Lys Thr Val Ile Arg Ser Ile Arg Ile Leu Lys Phe Leu Val 530 535 540 Ala Lys Arg Lys Phe Lys Glu Thr Leu Arg Pro Tyr Asp Val Lys Asp 545 550 555 560 Val Ile Glu Gln Tyr Ser Ala Gly His Leu Asp Met Leu Gly Arg Ile 565 570 575 Lys Ser Leu Gln Thr Arg Val Asp Gln Ile Val Gly Arg Gly Pro Gly 580 585 590 Asp Arg Lys Ala Arg Glu Lys Gly Asp Lys Gly Pro Ser Asp Ala Glu 595 600 605 Val Val Asp Glu Ile Ser Met Met Gly Arg Val Val Lys Val Glu Lys 610 615 620 Gln Val Gln Ser Ile Glu His Lys Leu Asp Leu Leu Leu Gly Phe Tyr 625 630 635 640 Ser Arg Cys Leu Arg Ser Gly Thr Ser Ala Ser Leu Gly Ala Val Gln 645 650 655 Val Pro Leu Phe Asp Pro Asp Ile Thr Ser Asp Tyr His Ser Pro Val 660 665 670 Asp His Glu Asp Ile Ser Val Ser Ala Gln Thr Leu Ser Ile Ser Arg 675 680 685 Ser Val Ser Thr Asn Met Asp 690 695

Claims (19)

A composition for diagnosing potassium channelopathy, comprising an agent for detecting a KCNQ4 (Voltage-Gated Potassium Channel Subunit Kv7.4) protein variant, a fragment thereof, or a polynucleotide encoding the protein variant,
The KCNQ4 protein variant is a variant (G319D) in which the 319th amino acid, glycine, is replaced with aspartic acid,
A composition wherein the potassium channelopathy is non-syndromic deafness.
The composition of claim 1, wherein the KCNQ4 protein comprises the amino acid sequence of SEQ ID NO: 1.
The composition according to claim 1, wherein the polynucleotide encoding the KCNQ4 protein variant includes a mutation in which G, the 956th base, is replaced with A.
The composition according to claim 1, wherein the polynucleotide encoding the KCNQ4 protein includes the base sequence of SEQ ID NO: 2.
The method according to claim 1, wherein the composition is capable of measuring the expression level of a KCNQ4 protein variant, a fragment thereof, or a polynucleotide encoding the same.
delete A kit for diagnosing potassium channelopathy, comprising the composition of any one of claims 1 to 5,
The kit, wherein the potassium channelopathy is non-syndromic deafness.
A method of providing information for the diagnosis or treatment of potassium channelopathy, comprising the step of detecting or measuring the expression level of a KCNQ4 protein variant, a fragment thereof, or a polynucleotide encoding the protein variant from a biological sample isolated from an individual,
The KCNQ4 protein variant is a variant in which the 319th amino acid, glycine, is replaced with aspartic acid,
The method of claim 1, wherein the potassium channelopathy is non-syndromic deafness.
The method of claim 8, wherein the method
When a KCNQ4 protein variant, a fragment thereof, or a polynucleotide encoding the protein variant is detected or expressed in the isolated biological sample, determining the individual as having developed potassium channelopathy or predicting the risk of developing it at a high level A method further comprising:
delete The method of claim 9, wherein the method
If the KCNQ4 protein variant is a heterozygous variant in which glycine, the 319th amino acid, is substituted with aspartic acid, the potassium channelopathy further includes the step of determining that it is caused by hyperactivation of the potassium channel function. , method.
delete The method of claim 9, wherein the method
If the KCNQ4 protein variant is a heterozygous variant in which glycine, the 319th amino acid, is substituted with aspartic acid, the method further comprises the step of determining the individual as a target for treatment using a potassium channel activity inhibitor.
A composition for screening potassium channel activity inhibitors, comprising a KCNQ4 wild-type protein and a transformant expressing a KCNQ4 protein variant in which glycine, the 319th amino acid, is substituted with aspartic acid.
The composition of claim 14, wherein the KCNQ4 wild-type protein and the KCNQ4 protein variant are expressed at a ratio of 7:1 to 1:2.
The composition according to claim 14, wherein the KCNQ4 wild-type protein and the KCNQ4 protein variant are linked to each other.
1) Treating a potassium channel activity inhibitor candidate to a transformant expressing the KCNQ4 wild-type protein and a KCNQ4 protein variant in which glycine, the 319th amino acid, is substituted with aspartic acid; and
2) Measuring the degree of potassium channel activation of the transformant treated with the candidate material
A method for screening potassium channel activity inhibitors, including a method.
The method of claim 17, wherein the method
The method further includes the step of determining that the candidate substance is a potassium channel activity inhibitor when activation of the potassium channel is inhibited by the candidate substance.
The method of claim 17, wherein the step of measuring the degree of potassium channel activation uses a patch clamp.