CN115820654A - LOXHD1 gene mutant and application thereof - Google Patents

Abstract

The invention provides a gene mutant related to non-syndromic deafness and application thereof. Compared with the wild LOXHD1 gene, the provided gene mutation has c.5331+1G >. The gene mutation is a new related pathogenic mutation of non-syndromic deafness, and whether the biological sample suffers from non-syndromic deafness can be effectively detected by detecting whether the mutation exists in the biological sample. The gene mutation further expands and perfects the detection and research of hereditary hearing loss diseases, and provides a new detection site, a new detection method and a new detection way for the diagnosis and treatment of the diseases.

Description

LOXHD1 gene mutant and its application

technical field

The present invention relates to the field of biology, in particular to gene mutants and applications thereof, more specifically to gene mutations, nucleic acids, polypeptides, biological models, drugs for treating non-syndromic deafness, and drugs for detecting non-syndromic deafness. Kits, constructs and recombinant cells.

Background technique

Deafness (hearing loss, HL) is the most common sensory dysfunction disease, and a considerable number of deafness patients are related to genetic factors. Determining the molecular mechanism of deafness through genetic testing, and then taking further prenatal genetic diagnosis and intervention measures, is an effective means to reduce the incidence of deafness, and it is also one of the fundamental ways to prevent and treat deafness. According to whether there are other concurrent clinical phenotypes, hereditary deafness can be divided into syndromic hearing loss (SHL) and non-syndromic hearing loss (NSHL).

Non-syndromic deafness means that deafness is the only symptom of the individual with no other genetic damage or organ dysfunction. Non-syndromic deafness accounts for about 70% of congenital hereditary deafness, of which 75%-80% are normal. Chromosomal recessive inheritance (autosomal recessive, AR). To date, more than 100 loci (loci) have been associated with autosomal recessive deafness, and these loci have been named in the form of DFNB+number. Non-syndromic deafness caused by different pathogenic genes has obvious differences in age of onset, degree of hearing loss, and progressiveness. Determining the causative gene of deafness will help to select appropriate hearing interventions for patients and better improve the quality of life of deaf patients.

With the development of sequencing technology, more and more genes related to hereditary deafness have been identified, which provides a basis for the molecular diagnosis of hereditary deafness and enables more hereditary deafness to be diagnosed and treated. However, due to the strong genetic heterogeneity of hereditary deafness, there are still a large number of pathogenic genes that have not been identified, so there is still a lot of room for research in this area, and research on gene identification still needs to be strengthened.

Contents of the invention

The present invention aims to solve one of the technical problems in the related art at least to a certain extent. Therefore, an object of the present invention is to provide a LOXHD1 gene mutant and its application.

LOXHD1 is the deafness-causing gene corresponding to the DFNB77 locus. The lipoxygenase homology domain 1 protein (lipoxygenase homology domain 1) encoded by this gene is mainly expressed in the hair cells of the cochlea and plays an important role in maintaining normal cell functions. Previous functional experiments in animal models have shown that individual mice carrying homozygous deleterious mutations will have an early-onset profound deafness phenotype.

Cases of deafness caused by pathogenic mutations in the LOXHD1 gene are extremely rare. So far, there have been only more than 30 family reports in the world. There are many phenotypic differences in patients in different reports, suggesting that there may be environmental factors or genetic regulatory mechanisms; in the reported cases, the pathogenicity of some mutations is not yet clear. Therefore, the research on autosomal recessive non-syndromic deafness related to the LOXHD1 gene still needs to be in-depth, and the discovery of new mutants will help further research on the corresponding pathogenic mechanism and clinical characteristics.

The inventor conducted a self-collected case of autosomal recessive non-syndromic deafness Trio pedigree (parents + proband), through the method of multi-gene capture sequencing Panel detection, family analysis combined with Sanger sequencing verification to detect and verify the pathogenic mutation; At the same time, the copy number variation that appears in the sample DNA is verified by qPCR method. Furthermore, the inventors discovered a new pathogenic site of the autosomal recessive non-synthetic deafness gene LOXHD1, which can be used for early screening of autosomal recessive non-synthetic deafness mutation carriers , and then carry out early intervention and treatment before the onset of the carrier; it can also be used for the molecular diagnosis of patients with autosomal recessive non-synthetic deafness and the differential diagnosis of related diseases, and it is fast, accurate, efficient, simple, and has a high early diagnosis rate. The results can provide a scientific basis for the early diagnosis and differential diagnosis of autosomal recessive non-synthetic deafness and the development of drugs for the treatment of autosomal recessive non-synthetic deafness.

In the first aspect of the present invention, the present invention provides a gene mutation, which has c.5331+1G>C mutation and/or c.1973_2437+2del mutation compared with the wild-type LOXHD1 gene. The inventors discovered for the first time that the c.5331+1G>C mutation and/or c.1973_2437+2del mutation of the LOXHD1 gene are closely related to the onset of non-syndromic deafness, so that by detecting whether the above gene mutation occurs in biological samples, it can be Efficient detection of non-syndromic deafness in biological samples.

In a second aspect of the present invention, the present invention provides a nucleic acid having a c.5331+1G>C mutation and/or a c.1973_2437+2del mutation compared to the wild-type LOXHD1 gene. The inventors discovered for the first time that the c.5331+1G>C mutation and/or c.1973_2437+2del mutation of the LOXHD1 gene are closely related to the onset of non-syndromic deafness, so that by detecting whether the nucleic acid exists in biological samples, it can be effectively To detect whether biological samples suffer from non-syndromic deafness.

In the third aspect of the present invention, the present invention provides a polypeptide expressed from the nucleic acid described in the second aspect of the present invention. Through research, it is found that the c.5331+1G>C mutation occurs on the LOXHD1 gene, which makes the produced mRNA unstable or easy to degrade, resulting in the synthesis of proteins that are different from the proteins or polypeptides expressed by the wild-type LOXHD1 gene or cause no protein expression or peptides. The polypeptide is closely related to the onset of non-syndromic deafness, so whether the polypeptide exists in the biological sample can be detected effectively to detect whether the biological sample suffers from non-syndromic deafness.

In the fourth aspect of the present invention, the present invention provides a use of a reagent for detecting gene mutations or nucleic acids or polypeptides in the preparation of kits or equipment for diagnosing non-syndromic deafness, said The gene mutation is the gene mutation described in the first aspect of the present invention, the nucleic acid is the nucleic acid described in the second aspect of the present invention, and the polypeptide is the polypeptide described in the third aspect of the present invention. As mentioned above, the above-mentioned gene mutations, nucleic acids, and polypeptides are closely related to the onset of non-syndromic deafness, and reagents capable of detecting the above-mentioned gene mutations, nucleic acids, or polypeptides can be used to prepare kits or equipment, The obtained kit or device can effectively screen biological samples suffering from non-syndromic deafness, especially autosomal recessive non-syndromic deafness.

In a fifth aspect of the present invention, the present invention provides a use of a biological model in drug screening, said biological model carrying at least one of the following: (1) the aforementioned gene mutation; (2) the aforementioned Nucleic acid; (3) expression of the aforementioned polypeptide. It should be noted that "the biological model carries the gene mutation described in the first aspect of the present invention" means that the LOXHD1 gene carried by the biological model of the present invention has c.5331+1G>C mutation and / or c.1973_2437+2del mutation; "the biological model carries the aforementioned nucleic acid" means that the biological model of the present invention carries the c.5331+1G>C mutation and/or the c.1973_2437+2del mutation compared with the LOXHD1 gene Nucleic acid sequence; "Biological model carrying the aforementioned polypeptide" means that the biological model of the present invention carries the c.5331+1G>C mutation and/or c.1973_2437+2del mutation of the LOXHD1 gene compared with the wild-type LOXHD1 gene the expressed polypeptide. The biological model thus provided can be effectively used as a model for the related research of non-syndromic deafness, especially autosomal recessive non-syndromic deafness.

In the sixth aspect of the present invention, the present invention provides the use of reagents for specifically changing gene mutations or nucleic acids in the preparation of medicines for the treatment of non-syndromic deafness, and the gene mutations are the first aspect of the present invention For the gene mutation, the nucleic acid is the nucleic acid described in the second aspect of the present invention. It should be noted that the specific change here refers to the ability to restore the mutated nucleic acid or gene mutation site to the original wild state or other non-pathogenic state without substantially affecting other sequences of the individual genome . As mentioned above, the above-mentioned nucleic acid or the above-mentioned gene mutation is closely related to the onset of non-syndromic deafness, especially autosomal recessive non-syndromic deafness, thus, specifically changing the above-mentioned The medicine prepared by the nucleic acid or the aforementioned gene mutation reagent can be effectively used to treat non-syndromic deafness, especially autosomal recessive non-syndromic deafness.

In the seventh aspect of the present invention, the present invention provides a medicine for the treatment of non-combined deafness, the medicine contains: specifically changing the gene mutation described in the first aspect of the present invention or the gene mutation described in the second aspect of the present invention nucleic acid reagents. It should be noted that the specific change here refers to the ability to restore the mutated nucleic acid or gene mutation site to the original wild state or other non-pathogenic state without substantially affecting other sequences of the individual genome . As mentioned above, the aforementioned nucleic acid or the aforementioned gene mutation is closely related to the onset of non-syndromic deafness. Drugs are effective in the treatment of non-syndromic deafness.

In the eighth aspect of the present invention, the present invention provides a construct comprising the gene mutation described in the first aspect of the present invention or the nucleic acid described in the second aspect of the present invention. It should be noted that "the construct comprises the gene mutation described in the first aspect of the present invention" means that the construct of the present invention has c.5331+1G>C mutation and/or c.1973_2437+compared with the wild-type LOXHD1 gene 2del mutation; "the construct comprises the nucleic acid described in the second aspect of the present invention" means that, compared with the wild-type LOXHD1 gene, the construct of the present invention contains the nucleic acid c.5331+1G>C mutation and/or c. 1973_2437+2del mutation. Thus, the recombinant cells obtained by transforming recipient cells with the constructs according to the embodiments of the present invention can be effectively used as models for related studies of non-syndromic deafness, especially autosomal recessive non-syndromic deafness.

In the ninth aspect of the present invention, the present invention provides a recombinant cell obtained by transforming a recipient cell with the construct described in the eighth aspect of the present invention or expressing the polypeptide described in the third aspect of the present invention of. The recombinant cell of the present invention can be effectively used as a model for related research on non-syndromic deafness, especially autosomal recessive non-syndromic deafness.

In the tenth aspect of the present invention, the present invention provides a kit for detecting non-syndromic deafness, which includes reagents for detecting the gene mutation described in the first aspect of the present invention, and/or detects the A reagent for the nucleic acid of the second aspect, and/or a reagent for detecting the polypeptide of the third aspect of the present invention. The aforementioned gene mutations, nucleic acids, and polypeptides are closely related to the onset of non-syndromic deafness, and can be used to contain reagents that can effectively detect the aforementioned nucleic acids or the aforementioned gene mutations or the aforementioned polypeptides The kit can effectively screen out biological samples suffering from non-syndromic deafness, especially autosomal recessive non-syndromic deafness.

Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, wherein:

Fig. 1 shows a Trio pedigree diagram of recessive genetic non-synthetic deafness provided according to an embodiment of the present invention.

Fig. 2 shows a graph of pure tone audiometry results of patients in the patient family shown in Fig. 1 according to an embodiment of the present invention.

Fig. 3 shows a representative Sanger sequencing verification peak map of the c.5331+1G>C mutation site of the LOXHD1 gene of all family members in the patient family shown in Fig. 1 provided according to an embodiment of the present invention.

Fig. 4 shows the results of qPCR verification of the LOXHD1 gene copy number variation of all family members in the patient family shown in Fig. 1 according to an embodiment of the present invention.

Detailed ways

The embodiments described below by referring to the figures are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

The mainstream method for the molecular diagnosis of hereditary deafness has changed from traditional single-gene sequencing to multi-gene capture sequencing Panel. There are more than 200 registered hereditary deafness multi-gene capture sequencing panels in the National Center for Biotechnology Information (NCBI) database, and the corresponding detection range includes dozens or even hundreds of deafness-related genes. Compared with single-gene sequencing, the multi-gene capture sequencing Panel method has a wide detection range and low unit cost, and can comprehensively screen a wide range of target regions at one time. At the same time, this method can also detect large deletions/duplications at the exon level, that is, copy number variants (CNVs), thereby covering more possible pathogenic mechanisms.

The inventor conducted a self-collected case of autosomal recessive non-syndromic deafness Trio pedigree (parents + proband), through the method of multi-gene capture sequencing Panel detection, family analysis combined with Sanger sequencing verification to detect and verify the pathogenic mutation; At the same time, the copy number variation that appears in the sample DNA is verified by qPCR method. Furthermore, the inventors discovered a new pathogenic site of the autosomal recessive non-synthetic deafness gene LOXHD1—the c.5331+1G>C mutation of the LOXHD1 gene. The mutation is located at the classic splicing position and has no records in multiple public databases. It is a strong low-frequency mutation; and the mutation forms a compound complex with the copy number variation of exon 15 to 17 deletion of the same gene on another chromosome of the patient. Combined (in trans), mutations occur at both sites, which will lead to the occurrence of autosomal recessive non-syndromic deafness. In addition, since these two sites are recessive mutations, when any site itself has a homozygous mutation, it will lead to the occurrence of non-syndromic deafness.

In this paper, the term "non-syndromic deafness" is also often referred to as "non-syndromic hereditary deafness" in the field, which means that deafness is the only symptom of an individual, not accompanied by other genetic damage and organ dysfunction .

The term "autosomal recessive non-syndromic deafness" is also commonly referred to as "autosomal recessive non-syndromic hereditary deafness" or "non-syndromic autosomal recessive deafness" or " "Non-syndromic autosomal recessive deafness" means that hereditary deafness is controlled by recessive alleles that occur on autosomes, that is, both alleles must be recessive for the patient to be Manifested as sick.

Moreover, it should be noted that the mutation site on the LOXHD1 gene provided herein can be used as a marker for non-syndromic deafness, more specifically autosomal recessive non-syndromic deafness; or these mutations The presence of a locus is used to indicate that the biological sample has non-syndromic deafness, more specifically autosomal recessive non-syndromic deafness. This does not mean that "non-syndromic deafness" and "autosomal recessive non-syndromic deafness" are a limitation of the mutation site on the LOXHD1 gene. That is to say, if the disease characterized by the mutation site on the LOXHD1 gene is to be indicated or explained in detail, it can be known that it can indicate that the biological sample is suffering from non-syndromic deafness, more specifically, normal deafness. Chromosomal recessive non-syndromic deafness; but it is also possible to be directly informed of hereditary deafness, or deafness, according to specific purposes or different targets.

In this paper, information about the wild-type LOXHD1 gene such as the DNA sequence (for example, including intron sequence, exon sequence, etc.), RNA sequence, and encoded protein information of the wild-type LOXHD1 gene is included in the NCBI database. It can be obtained by referring to the following URL: https://www.ncbi.nlm.nih.gov/gene/125336. The c.5331+1G>C mutation and c.1973_2437+2del mutation shown in this article were determined based on the sequence of the wild-type LOXHD1 gene in the NCBI database as a reference.

Herein, the shown c.5331+1G>C mutation refers to the mutation of the first base G to C in intron 34 of the wild-type LOXHD1 gene.

At the same time, for the convenience of viewing, the partial nucleic acid sequence of the wild-type LOXHD1 gene is provided as follows, the c.5331+1G>C mutation corresponds to the 507th base G mutation of the part of the wild-type LOXHD1 gene sequence (SEQ ID NO: 1) as C, that is, the bolded and boxed base G in SEQ ID NO: 1 below will be mutated into base C:

The coding DNA of this part of the wild-type LOXHD1 gene (corresponding to the NCBI database number is NCBI ReferenceSequence: NG_016646.2) has the following nucleotide sequence (SEQ ID NO: 1) from 151831 to 151230, wherein in SEQ ID NO: 1 Underlined bold bases are the matching positions of primers SEQ ID NO:7 and SEQ ID NO:8:

Compared with the wild-type LOXHD1 gene, the LOXHD1 gene mutant discovered by the inventor has a c.5331+1G>C mutation, that is, compared with the wild-type LOXHD1 gene, the first base of intron 34 of the LOXHD1 mutant G is mutated to C, corresponding to the 507th base G in the above reference sequence (SEQ ID NO: 1) is mutated to C. Therefore, compared with the wild-type product, the donor site (donor site) of the classical splicing position on intron 34 is destroyed, thereby affecting normal splicing.

The above c.5331+1G>C mutation and the deletion of exons 15 to 17 of the same gene on another chromosome (NG_016646.2(LOXHD1_v001):c.1973_2437+2del) copy number variation constitute compound heterozygous (in trans ), leading to the occurrence of autosomal recessive non-syndromic deafness. The "copy number variation due to deletion of exon 15 to 17" here refers to the deletion of the sequence from exon 15 to 17, resulting in copy number variation due to copy number reduction, expressed as c.1973_2437+2del mutation.

At the same time, for the convenience of viewing, the part of the wild-type LOXHD1 gene sequence corresponding to the c.1973_2437+2del mutation is provided as follows. The c.1973_2437+2del mutation corresponds to the part of the wild-type LOXHD1 gene sequence (SEQ ID NO: 2) No. This sequence of exon No. is deleted, that is, the underlined sequence in the following SEQ ID NO:2 is deleted:

The nucleic acid sequence of this part of the wild-type LOXHD1 gene (corresponding to NCBI database number: NG_016646.2) has the following nucleotide sequence (SEQ ID NO: 2) from 89601 to 95900, and the bold base is the primer SEQ ID NO :3～primer SEQ ID NO:6 matching position:

It should be noted that the mutation sites and sequences given above are all based on the contents recorded in the NCBI database as a reference. Those skilled in the art should understand that due to the update of the database or the difference in the database, the The mutation site and sequence may be slightly different or changed, and these differences or changes can be found in the given database as a standard, and these differences or changes are also included in the protection scope of the present invention.

Moreover, those skilled in the art can understand that the sequence position of the wild-type LOXHD1 gene used herein is based on the sequence of the wild-type LOXHD1 gene in the human genome, but when the wild-type LOXHD1 gene exists in other species, the The sequence may be different, and the wild-type LOXHD1 gene of the species can be compared with the wild-type LOXHD1 gene in the human genome to obtain the corresponding position in the wild-type LOXHD1 gene of the species.

Gene mutation

In one aspect of the invention, the invention proposes a genetic mutation. According to an embodiment of the present invention, compared with the wild-type LOXHD1 gene, it has a c.5331+1G>C mutation and/or a c.1973_2437+2del mutation. The inventors found that the c.5331+1G>C mutation and/or the c.1973_2437+2del mutation of the LOXHD1 gene are closely related to the onset of non-syndromic deafness (such as autosomal recessive non-syndromic deafness), so that by detecting the above-mentioned genes Whether the mutation occurs in the biological sample can effectively detect whether the biological sample suffers from non-syndromic deafness.

Gene mutation usually refers to the change of base pair composition or sequence in gene structure. Herein, the gene mutation refers to a mutation occurring in the LOXHD1 gene. The gene mutation can be detected or identified. It can be detected or identified as a mutation site on the LOXHD1 gene, or it can be detected or identified as a part of the nucleic acid of the LOXHD1 gene or the entire nucleic acid of the LOXHD1 gene. distinguish. Of course, it can also be said that the gene mutation can be selected. Antibodies, probes, primers, and mass spectrometry detection reagents commonly used in the art can be used to detect the above gene mutations.

nucleic acid

In yet another aspect of the present invention, the present invention provides a nucleic acid. According to an embodiment of the present invention, compared with the wild-type LOXHD1 gene, the nucleic acid has the following mutations: c.5331+1G>C mutation and/or c.1973_2437+2del mutation. The inventors found that the c.5331+1G>C mutation and/or the c.1973_2437+2del mutation of the LOXHD1 gene are closely related to the onset of non-syndromic deafness, so by detecting whether the above nucleic acid exists in biological samples, it can effectively detect Whether the biological sample suffers from non-syndromic deafness.

As for nucleic acid mentioned in the specification and claims of the present invention, those skilled in the art should understand that it actually includes any one or both of the complementary double strands. For convenience, in this specification and claims, although only one chain is given in most cases, another chain complementary to it is actually disclosed. For example, reference to the sequence of the LOXHD1 gene actually includes its complement. It will also be understood by those skilled in the art that one strand can be used to detect the other, and vice versa.

It should be noted that the coding sequence of the wild-type LOXHD1 gene can be obtained from the following website: https://www.ncbi.nlm.nih.gov/gene/125336.

The c.5331+1G>C mutation and/or c.1973_2437+2del mutation mentioned in this article are located in the coding sequence corresponding to the above website.

polypeptide

In another aspect of the present invention, the present invention provides a polypeptide. According to an embodiment of the present invention, the polypeptide is expressed from the above nucleic acid. As mentioned above, the c.5331+1G>C mutation and/or c.1973_2437+2del mutation of the LOXHD1 gene is closely related to the onset of non-syndromic deafness, and the protein expressed by the mutant gene is similar to that of non-syndromic deafness. The pathogenesis is closely related, and by detecting whether the above polypeptide exists in the biological sample, it can effectively detect whether the biological sample suffers from non-syndromic deafness.

Use of reagents for detecting nucleic acids, gene mutations, and polypeptides described above in the preparation of kits or equipment

In yet another aspect of the present invention, the present invention proposes the use of the above-mentioned gene mutation or the above-mentioned nucleic acid or the above-mentioned polypeptide reagent in the preparation of kits or equipment. According to an embodiment of the present invention, the kit or device is used for diagnosing non-syndromic deafness. As mentioned above, the aforementioned nucleic acid, gene mutation, and polypeptide are closely related to the onset of non-syndromic deafness, and then the reagents for detecting the aforementioned nucleic acid or the aforementioned gene mutation or the aforementioned polypeptide are used for A kit or device is prepared, and the obtained kit or device can effectively screen biological samples suffering from non-syndromic deafness, especially autosomal recessive non-syndromic deafness.

According to an embodiment of the present invention, the non-syndromic deafness is autosomal recessive non-syndromic deafness.

According to an embodiment of the present invention, the reagent includes at least one of an antibody, a probe, a primer and a mass spectrometry detection reagent specific for at least one of the nucleic acid, the gene mutation and the polypeptide. For example, the inventor can detect whether the above-mentioned mutation exists in the sample to be tested through the specific binding of the antibody that specifically recognizes the polypeptide and the polypeptide, that is, detect the presence of the above-mentioned polypeptide through the interaction between the specific antibody and the antigen; The inventor can also identify the presence of the above-mentioned nucleic acid or gene mutation by pre-designing a probe that specifically recognizes the nucleic acid or gene mutation, and through complementary pairing between the probe and the nucleic acid fragment where the above-mentioned nucleic acid or gene mutation site is located; People can also design specific primers for amplifying the exon where the above-mentioned gene mutation is located, and then determine whether the above-mentioned gene mutation exists through gene amplification and sequencing; the inventor can also use mass spectrometry to detect the m/z of the polypeptide to judge , Whether the polypeptide expressed by the c.5331+1G>C mutation and/or c.1973_2437+2del mutation exists. At least one of the antibodies, probes, primers and mass spectrometry detection reagents according to specific embodiments of the present invention can specifically and highly sensitively screen out the aforementioned nucleic acid or the aforementioned gene mutation or the aforementioned polypeptide , and then specifically and highly sensitively screen out biological samples suffering from non-syndromic deafness, especially those suffering from autosomal recessive non-syndromic deafness, which can be effectively used to prepare and screen for non-syndromic deafness, especially Kit or device for biological samples of autosomal recessive non-syndromic deafness.

biological model

In another aspect of the present invention, the present invention proposes the use of biological models in screening drugs. According to an embodiment of the present invention, the biological model carries at least one of the following: (1) the aforementioned nucleic acid; (2) the aforementioned gene mutation; (3) expressing the aforementioned polypeptide. It should be noted that "the biological model carries the aforementioned nucleic acid" means that the biological model of the present invention carries the LOXHD1 with c.5331+1G>C mutation and/or c.1973_2437+2del mutation compared with the wild-type LOXHD1 gene The nucleic acid sequence of the gene mutant; "the biological model carries the aforementioned gene mutation" means that the LOXHD1 gene carried by the biological model of the present invention has c.5331+1G>C mutation and/or c. 1973_2437+2del mutation. "The biological model carries the aforementioned polypeptide" means that the biological model of the present invention carries the polypeptide expressed by the c.5331+1G>C mutation and/or the c.1973_2437+2del mutation compared with the wild-type LOXHD1 gene. The biological model according to the embodiment of the present invention can be effectively used as a model for related studies of non-syndromic deafness, especially autosomal recessive non-syndromic deafness. These biological models can be cell models or animal models.

The use of reagents in the preparation of medicines

In yet another aspect of the present invention, the present invention proposes the use of reagents for specifically changing the above-mentioned nucleic acid or the above-mentioned gene mutation in the preparation of medicines for the treatment of non-syndromic deafness. It should be noted that the specific change here refers to the ability to restore the mutated nucleic acid or gene mutation site to the original wild state or other non-pathogenic state without substantially affecting other sequences of the individual genome . As mentioned above, the above-mentioned nucleic acid or the above-mentioned gene mutation is closely related to the onset of non-syndromic deafness. The medicine prepared by the reagent can be effectively used for treating non-syndromic deafness.

According to an embodiment of the present invention, the reagent is a reagent based on at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease. For example, CRISPRs technology is a technology that guides Cas proteins to modify targeted genes by RNA. CRISPR-Cas9 mainly realizes genome modification through gene knockout, introduction of special mutations and site-specific transgenesis. CRISPR-Cas9-based Method, the inventor can design sgRNA and synthesize the gRNA of this sequence, and then co-express the gRNA and dCas9 in the cell, and use the gRNA to mediate the binding of the dCas9 protein to the target DNA region, thereby realizing the repair or change of a specific site.

Drugs for the treatment of nonsyndromic deafness

In another aspect of the present invention, the present invention proposes a medicine for treating non-syndromic deafness. According to an embodiment of the present invention, the medicine contains: a reagent for specifically changing the aforementioned nucleic acid or the aforementioned gene mutation. It should be noted that the specific change here refers to the ability to restore the mutated nucleic acid or gene mutation site to the original wild state or other non-pathogenic state without substantially affecting other sequences of the individual genome . As mentioned above, the above-mentioned nucleic acid or the above-mentioned gene mutation is closely related to the onset of non-syndromic deafness, especially the onset of autosomal recessive non-syndromic deafness, thus, including the specific changes mentioned above The nucleic acid or the drug of the aforementioned gene mutation agent can be effectively used for the treatment of non-syndromic deafness.

According to an embodiment of the present invention, the reagent is a reagent based on at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease. For example, CRISPRs technology is a technology that guides Cas proteins to modify targeted genes by RNA. CRISPR-Cas9 mainly realizes genome modification through gene knockout, introduction of special mutations and site-specific transgenesis. CRISPR-Cas9-based Method, the inventor can design sgRNA and synthesize the gRNA of this sequence, and then co-express the gRNA and dCas9 in the cell, and use the gRNA to mediate the binding of the dCas9 protein to the target DNA region, thereby realizing the repair or change of a specific site.

Constructs and Recombinant Cells

In yet another aspect of the invention, the invention proposes a construct. According to an embodiment of the present invention, the construct comprises the aforementioned nucleic acid or the aforementioned gene mutation. It should be noted that "the construct comprises the aforementioned nucleic acid" means that the construct of the present invention comprises, compared with the wild-type LOXHD1 gene, a gene with c.5331+1G>C mutation and/or c.1973_2437+2del mutation nucleic acid. "The construct comprises the aforementioned gene mutation" means that the construct of the present invention has c.5331+1G>C mutation and/or c.1973_2437+2del mutation compared with the wild-type LOXHD1 gene. Thus, the recombinant cells obtained by transforming recipient cells with the constructs according to the embodiments of the present invention can be effectively used as models for related studies of non-syndromic deafness, especially autosomal recessive non-syndromic deafness. Wherein, the type of the recipient cells is not particularly limited, for example, Escherichia coli cells, mammalian cells, preferably the recipient cells are derived from mammals.

The term "construct" used in the present invention refers to a genetic carrier that contains a specific nucleic acid sequence and is capable of transferring the target nucleic acid sequence into a host cell to obtain a recombinant cell. According to the embodiments of the present invention, the form of the construct is not particularly limited. According to an embodiment of the present invention, it may be at least one of a plasmid, a phage, an artificial chromosome, a cosmid (Cosmid), and a virus, preferably a plasmid. As a genetic carrier, plasmids are easy to operate and can carry large fragments, which is convenient for operation and processing. The form of the plasmid is also not particularly limited, and it can be either a circular plasmid or a linear plasmid, that is, it can be single-stranded or double-stranded. Those skilled in the art can make selections as needed. The term "nucleic acid" used in the present invention can be any polymer comprising deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, its length is not subject to any Special restrictions. For constructs for constructing recombinant cells, the nucleic acid is preferably DNA, because DNA is more stable and easier to handle than RNA.

In yet another aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell is obtained by transforming the recipient cell with the aforementioned construct or expressing the aforementioned polypeptide. According to some embodiments of the present invention, the recombinant cells of the present invention can be effectively used as a model for related studies on non-syndromic deafness, especially autosomal recessive non-syndromic deafness.

According to the embodiment of the present invention, the type of recipient cells is not particularly limited, for example, it can be Escherichia coli cells, mammalian cells, preferably the recipient cells are derived from non-human mammals.

Kit for detecting non-syndromic deafness

In another aspect of the present invention, the present invention provides a kit for detecting non-syndromic deafness. According to an embodiment of the present invention, the kit includes a reagent for detecting the aforementioned nucleic acid, and/or a reagent for detecting the aforementioned gene mutation, and/or a reagent for detecting the aforementioned polypeptide. As mentioned above, the aforementioned nucleic acid, gene mutation, and polypeptide are closely related to the onset of non-syndromic deafness, and can be used to effectively detect the aforementioned nucleic acid or the aforementioned gene mutation or the aforementioned The reagent kit of the polypeptide can effectively screen out biological samples suffering from non-syndromic deafness, especially autosomal recessive non-syndromic deafness.

Method for screening biological samples suffering from non-syndromic deafness

In addition to the above, the present invention also proposes a method for screening biological samples suffering from non-syndromic deafness. According to an embodiment of the present invention, the method includes the following steps:

extraction of nucleic acid samples from biological samples;

determining the nucleic acid sequence of the nucleic acid sample based on the nucleic acid sample;

Based on the nucleic acid sequence of the nucleic acid sample or its complementary sequence, compared with the wild-type LOXHD1 gene, whether it has c.5331+1G>C mutation and/or c.1973_2437+2del mutation, to determine whether the biological sample is suffering from non-syndromic Type deafness, wherein, the nucleic acid sequence of the nucleic acid sample or its complementary sequence has c.5331+1G>C mutation and/or c.1973_2437+2del mutation compared with the wild-type LOXHD1 gene, which means that the biological sample suffers from Indication of non-syndromic deafness. Through the method for screening biological samples suffering from non-syndromic deafness according to the embodiments of the present invention, biological samples suffering from non-syndromic deafness, especially autosomal recessive non-syndromic deafness, can be effectively screened.

First, a nucleic acid sample is extracted from a biological sample. According to the embodiments of the present invention, the type of biological sample is not particularly limited, as long as a nucleic acid sample reflecting whether there is a mutation in the LOXHD1 gene of the biological sample can be extracted from the biological sample. According to an embodiment of the present invention, the biological sample may be at least one selected from human blood, skin, and subcutaneous tissue. Thus, sampling and detection can be conveniently performed, thereby further improving the efficiency of screening biological samples suffering from non-syndromic deafness. According to an embodiment of the present invention, the term "nucleic acid sample" used here should be understood in a broad sense, and it can be any sample that can reflect whether there is a mutation in the LOXHD1 gene in a biological sample, for example, it can be a whole genome directly extracted from a biological sample The DNA may also be a part of the whole genome containing the coding sequence of the LOXHD1 gene, or it may be the total RNA extracted from the biological sample, or it may be the mRNA extracted from the biological sample. According to an embodiment of the present invention, the nucleic acid sample is whole genome DNA. Thus, the range of sources of biological samples can be expanded, and multiple information of biological samples can be determined simultaneously, thereby improving the efficiency of screening biological samples suffering from non-syndromic deafness. In addition, according to an embodiment of the present invention, for using RNA as a nucleic acid sample, extracting a nucleic acid sample from a biological sample may further include: extracting an RNA sample from a biological sample, preferably the RNA sample is mRNA; and based on the obtained RNA sample, by inversion The reaction is recorded to obtain a cDNA sample, and the obtained cDNA sample constitutes a nucleic acid sample. Thus, the efficiency of using RNA as a nucleic acid sample to screen biological samples suffering from non-syndromic deafness can be further improved.

Next, after the nucleic acid sample is obtained, the nucleic acid sample can be analyzed, so that the nucleic acid sequence of the obtained nucleic acid sample can be determined. According to the embodiments of the present invention, the method and device for determining the nucleic acid sequence of the obtained nucleic acid sample are not particularly limited. According to a specific embodiment of the present invention, the nucleic acid sequence of the nucleic acid sample can be determined by a sequencing method. According to the embodiments of the present invention, the methods and devices that can be used for sequencing are not particularly limited. According to the embodiment of the present invention, the second-generation sequencing technology may be used, and the third-generation and fourth-generation or more advanced sequencing technologies may also be used.

According to a specific embodiment of the present invention, the nucleic acid sequence can be sequenced by using at least one of BGISEQ-500, BGISEQ-500RS, HISEQ2000, SOLiD, 454 and single-molecule sequencing devices. Therefore, combined with the latest sequencing technology, a higher sequencing depth can be achieved for a single site, and the detection sensitivity and accuracy are greatly improved. Therefore, the high-throughput and deep sequencing characteristics of these sequencing devices can be used to further improve the detection of nucleic acid samples. Efficiency in conducting assays. Therefore, the precision and accuracy of the subsequent analysis of the sequencing data can be improved. Thus, according to an embodiment of the present invention, determining the nucleic acid sequence of the nucleic acid sample may further include: first, constructing a nucleic acid sequencing library for the obtained nucleic acid sample; and sequencing the obtained nucleic acid sequencing library, so as to obtain multiple Sequencing results composed of sequencing data. According to some embodiments of the present invention, at least one selected from BGISEQ-500, BGISEQ-500RS, HISEQ2000, SOLiD, 454, and single-molecule sequencing devices can be used to sequence the obtained nucleic acid sequencing library. In addition, according to the embodiments of the present invention, the nucleic acid samples can be screened to enrich the exons of the LOXHD1 gene, and the screening and enrichment can be performed before, during, or after the construction of the sequencing library. Exon-targeted sequence enrichment systems such as BGI's own exon capture chip, Aglient SureSelect, Nimblegen and other exon or target region capture platforms can be used to enrich target fragments. According to an embodiment of the present invention, for nucleic acid samples, constructing a nucleic acid sequencing library further includes: using at least one selected from the LOXHD1 gene-specific primers, performing PCR amplification on the nucleic acid samples; and for the obtained amplification products, constructing Nucleic acid sequencing library. Thus, the exons of the LOXHD1 gene can be enriched by PCR amplification, thereby further improving the efficiency of screening biological samples suffering from non-syndromic deafness. According to an embodiment of the present invention, the sequence of the LOXHD1 gene-specific primer is not particularly limited. For example, it can be obtained by referring to the human genome sequence database GRCh37.1/hg19 and using Primer3.0 online design. .ucsc.edu/), using Primer3 (version 0.4.0, http://primer3.ut.ee/) to design and synthesize primers for candidate genes (synthesized by Sangon Bioengineering Company), and using Primer-BLAST (http: http://www.ncbi.nlm.nih.gov/tools/primer-blast/) to verify primer specificity.

Regarding the method and process for constructing a sequencing library for nucleic acid samples, those skilled in the art can make appropriate choices according to different sequencing technologies. For details on the process, refer to the procedures provided by manufacturers of sequencing instruments such as Illumina, for example, see Illumina Multiplexing Sample Preparation Guide (Part#1005361; Feb 2010) or Paired-End SamplePrep Guide (Part#1005063; Feb 2010), which are incorporated herein by reference. According to the embodiments of the present invention, the method and equipment for extracting nucleic acid samples from biological samples are not particularly limited, and commercial nucleic acid extraction kits can be used.

It should be noted that the term "nucleic acid sequence" used here should be understood in a broad sense. It can be the complete nucleic acid sequence information obtained after the sequencing data obtained by sequencing nucleic acid samples are assembled, or it can be obtained directly. The sequencing data (reads) obtained by sequencing the nucleic acid samples are used as the nucleic acid sequences, as long as the nucleic acid sequences contain the coding sequence corresponding to the LOXHD1 gene.

Finally, after determining the nucleic acid sequence of the nucleic acid sample, compare the corresponding reference sequence of the obtained nucleic acid sample nucleic acid sequence, when the obtained nucleic acid sequence has the aforementioned mutation, it indicates that the biological sample suffers from non-syndromic deafness . Thus, biological samples suffering from non-syndromic deafness can be effectively screened through the method for screening biological samples suffering from non-syndromic deafness according to an embodiment of the present invention. According to the embodiments of the present invention, the method and equipment for comparing the nucleic acid sequence with the corresponding wild-type gene sequence are not particularly limited, and any conventional software can be used for operation. According to specific examples of the present invention, SOAPALIGNER/SOAP2 can be used Compare.

It should be noted that the use of the "method for screening biological samples suffering from non-syndromic deafness" according to the embodiment of the present invention is not particularly limited, for example, it can be used as a screening method for non-diagnostic purposes, such as for scientific research or other applications .

The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Example 1 Determining the causative mutation of autosomal recessive non-syndromic deafness

1. Sample collection

The inventor collected a Trio family (parents + proband) of a Chinese Han patient with autosomal recessive non-syndromic deafness. The pedigree is shown in FIG. 1 . Among them, ○ indicates a normal female, □ indicates a normal male, ● indicates a female patient, and the arrow points to the proband.

As shown in Figure 1, the family includes 3 members, the parents are normal (i.e., I-1 and I-2 in the pedigree diagram), and the daughter is a deaf patient (i.e., II-1 in the pedigree diagram). Chromosomal recessive inheritance pattern.

The pure tone audiometry results of the patients in this family are shown in Figure 2. In Figure 2, the abscissa indicates the frequency of the pure tone, and the ordinate indicates the hearing level. If the hearing is normal, the threshold curve should be floating around 0, and the curve going down indicates hearing loss. As shown in Figure 2, the results of pure tone audiometry showed that patient II-1 had moderate to severe deafness in the left ear and moderate deafness in the right ear, and the hearing curve showed a steep drop-off type at high frequencies.

The inventor collected peripheral blood samples of all members of the family, added EDTA for anticoagulation, and stored them at -80°C. All blood samples were signed informed consent.

2. DNA extraction

The peripheral blood of all members of the above-mentioned families was taken, and the genomic DNA of peripheral blood leukocytes was extracted by QIAmp Bloodkit (Qiagen, Hilden, Germany), and the concentration and purity of the DNA were measured by QubitFluorometer and agarose gel electrophoresis. Each sample obtained Genomic DNA OD260/OD280 are both between 1.7-2.0, the concentration is not less than 50 ng/μl, and the total amount is not less than 3 μg.

3. Capture sequencing

Using chip capture technology combined with Illumina Hiseq 2500 high-throughput sequencing technology, the samples of all family members were sequenced. The capture range of the corresponding custom capture chip (Agilent, Santa Clara, CA, USA) includes 127 known deafness-related genes, a total of 2268 exons and adjacent regions, and the total length of the capture region is 619Kb. The main steps include ultrasonic fragmentation, library preparation and on-machine sequencing.

After the sequencing data is off the machine, the internal customized process is used for variation detection, annotation, and database comparison, and candidate pathogenic loci are determined based on population frequency, software prediction results, and family analysis. At the same time, manually check the regions where the sequencing depth deviates from the normal range, and use qPCR to verify the suspected copy number variation.

Results A heterozygous c.5331+1G>C mutation was found in the patient's LOXHD1 gene. At the same time, the depth of exon 15 to exon 17 of the gene was abnormal, which was suspected to be a copy number variation of heterozygous deletion. Family analysis showed that the patient's mother and father were heterozygous for the c.5331+1G>C mutation and a suspected copy number variation in the same region, respectively.

According to the recessive inheritance model of LOXHD1 gene-related deafness, if the copy number variation is verified as true positive by qPCR, and the c. The cause of the patient's deafness.

4. qPCR verification of copy number variation

Design and synthesize specific primers based on exon 15 and exon 17 at both ends of the suspected heterozygous deletion region. The primer information is as follows:

Exon 15 upstream primer: TGATTGTCTGCCTGCTCCAC (SEQ ID NO: 3)

Downstream primer of exon 15: GCTGTCACTGGGTAGCAACT (SEQ ID NO: 4)

The upstream primer of exon 17: GTGATTGGGCATGACAGCAC (SEQ ID NO:5)

Downstream primer of exon 17: GTTGGCGGGAAAGGTGTACT (SEQ ID NO: 6)

The above-mentioned specific primers were used for qPCR verification, and the Power SYBR Green PCR MasterMix (ABI) was used to connect to the real-time system to quantify the data volume of the amplification primers. As shown in Figure 4, the quantitative results showed that compared with the normal control samples, the copy number variation of heterozygous deletion was confirmed in the exon 15 to 17 region of the LOXHD1 gene of the patient and father. Example 2 Sanger method sequencing verification

All family members (including patients and parents with normal hearing) in the family of patients with autosomal recessive non-synthetic deafness described in Example 1 were detected for the LOXHD1 gene: for the c.5331+1G>C mutation of the LOXHD1 gene Design primers, then obtain the relevant sequence of the mutation site by PCR amplification, product purification and sequencing, and verify whether the c.5331+1G>C mutation of the LOXHD1 gene is in the sample according to whether the sequence determination result is mutant or wild type Check out.

Specific steps are as follows:

1. DNA extraction

According to the DNA extraction method described in Example 1, the genomic DNA in the peripheral venous blood of the subject was extracted for future use.

2. Primer design and PCR reaction

First, referring to the human genome reference sequence GRCh37/hg19, specific primers for the c.5331+1G>C mutation of the LOXHD1 gene were designed, and the specific sequences were as follows:

Upstream primer: GGTGGAGAGTTGCTGAGAGC (SEQ ID NO: 7)

Downstream primer: CTGAAGGAAGCCCACACCAC (SEQ ID NO: 8)

Then, prepare the PCR reaction system of each DNA sample according to the following ratio and perform the PCR reaction:

Reaction system (20μl):

Reagent name Single reaction standard volume (μL) ddH₂O 17.30 10×Buffer 2.50 dNTP (2.5mM) 2.00 Primer-F (10p) 1.00 Primer-R (10p) 1.00 Taq enzyme (5U/q) 0.20 Total twenty four

PCR reaction conditions

Thus, the PCR amplification products of the genomic DNA samples of each subject were obtained.

3. Sanger sequencing

After the PCR product obtained in step 2 was purified, it was directly subjected to DNA sequencing, and the sequencing was performed using an ABI3730XL sequencer.

Based on the sequencing results, Figure 3 shows the representative Sanger sequencing verification peaks of the c.5331+1G>C mutation site in the LOXHD1 gene of all family members in the patient family shown in Figure 1. As shown in Figure 3, in the Trio family of patients with autosomal recessive non-syndromic deafness of the present invention, both the patient and the mother with normal hearing are heterozygous carriers of the c.5331+1G>C mutation of the LOXHD1 gene, and the father of the patient It is homozygous wild type, indicating that the patient's mutation was derived from the mother.

Figure 4 shows the results of qPCR verification of the LOXHD1 gene copy number variation of all family members in the patient family shown in Figure 1. The ordinate in Fig. 4 represents the relative quantification (relative quantification, RQ) result of qPCR. As shown in Figure 4, the RQ value of the sample corresponding to the patient and the father with normal hearing is half of that of the control sample, indicating that the two carry the copy number variation of the deletion in the region from exon 1115 to exon 17 of LOXHD, and the copy number of the corresponding sample of the patient’s mother The above results indicate that the copy number variation of the patient originated from the father with normal hearing.

Combining the above information, it can be confirmed that the compound heterozygous genotype formed by the LOXHD1 c.5331+1G>C mutation and another copy number variation is the cause of deafness patient II-1 in this recessive non-syndromic deafness family.

Embodiment 3 detection kit

A detection kit is prepared, which comprises primers capable of detecting the c.5331+1G>C mutation of the LOXHD1 gene, and is used for screening biological samples susceptible to autosomal recessive non-synthetic deafness. The corresponding primers are LOXHD1 gene exon-specific primers, the sequences of which are shown in SEQ ID NO:7 and SEQ ID NO:8 in Example 2.

The specific steps for screening biological samples susceptible to autosomal recessive non-synthetic deafness using the above kit are as follows:

According to the method described in step 2 of Example 1, the DNA of the test subject was extracted, and the PCR reaction was carried out with the extracted DNA as a template and the exon-specific primers of the above-mentioned LOXHD1 gene (see Example 2 for the PCR reaction system and reaction conditions, The PCR product was purified according to conventional methods in the art, and the purified product was sequenced.

Then by observing whether the sequence obtained by sequencing has the c.5331+1G>C mutation, it can effectively detect whether the LOXHD1 gene mutant of the present invention exists in the DNA of the test subject, thereby effectively detecting whether the test subject is susceptible to Autosomal recessive non-synthetic deafness, and further, biological samples susceptible to autosomal recessive non-synthetic deafness can be screened out from the test subjects.

Further, the primer sequence of SEQ ID NO: 3 to SEQ ID NO: 6 can also be used in the kit to specifically amplify the regions of exon 15 and exon 17 of the LOXHD1 gene for screening susceptible non-synthetic Biological samples of type deafness.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, those skilled in the art can make the above-mentioned The embodiments are subject to changes, modifications, substitutions and variations.

Claims (10)

1. A nucleic acid, characterized in that, compared with the wild-type LOXHD1 gene, the nucleic acid has a c.5331+1G>C mutation and/or a c.1973_2437+2del mutation. 2. A polypeptide, characterized in that, the polypeptide is expressed from the nucleic acid according to claim 1. 3. A detectable gene mutation, characterized in that, compared with the wild-type LOXHD1 gene, it has c.5331+1G>C mutation and/or c.1973_2437+2del mutation. 4. Use of the reagent for detecting the nucleic acid as claimed in claim 1 or the polypeptide as claimed in claim 2 or the gene mutation as claimed in claim 3 in the preparation of kits or equipment for diagnosing non-comprehensive Symptomatic deafness; Optionally, the non-syndromic deafness is autosomal recessive non-syndromic deafness; Optionally, the reagents include at least one of antibodies, probes, primers and mass spectrometry detection reagents specific for at least one of the gene mutation, the nucleic acid and the polypeptide. 5. The use of a biological model in screening drugs, the biological model carrying at least one of the following: (1) nucleic acid according to claim 1; (2) expressing the polypeptide according to claim 2; (3) the genetic mutation as claimed in claim 3; Optionally, the biological model is a cell model or an animal model. 6. The use of the reagent for specifically changing the nucleic acid according to claim 1 or the genetic mutation according to claim 3 in the preparation of medicines, which are used to treat non-syndromic deafness; Optionally, the non-syndromic deafness is autosomal recessive non-syndromic deafness; Optionally, the reagent is a reagent based on at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease. 7. A medicine for the treatment of non-comprehensive deafness, said medicine containing: A reagent for specifically changing the nucleic acid of claim 1 or the gene mutation of claim 3; Optionally, the non-syndromic deafness is autosomal recessive non-syndromic deafness; Optionally, the reagent is a reagent based on at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease. 8. A construct, characterized in that it comprises the nucleic acid of claim 1 or the gene mutation of claim 3. 9. A recombinant cell, characterized in that the recombinant cell is obtained by transforming a recipient cell with the construct of claim 8 or expressing the polypeptide of claim 2. 10. A test kit for detecting non-syndromic deafness, characterized in that, the test kit includes a reagent for detecting the nucleic acid according to claim 1, and/or a reagent for detecting the polypeptide according to claim 2, and /or a reagent for detecting the gene mutation described in claim 3.

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