Application of RGS12 gene in detection of deafness

Abstract

The present application relates to the field of biomedical technology, and particularly relates to the application of RGS12 gene in deafness detection. The present application fully proves that RGS12 gene is a new autosomal recessive deafness gene from animal models and deafness population samples, and can be used as a new target for deafness gene diagnosis. The discovery of the site will improve the detection rate in the process of clinical deafness gene screening. The present application provides a new theoretical basis for revealing the mechanism of deafness, and lays a foundation for developing an early warning model suitable for deafness disease, and therefore has good practical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of the RGS12 gene in deafness detection. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Deafness is one of the most common birth defects in humans, with an incidence rate of approximately 1-3 per 1,000 newborns. According to the World Health Organization (WHO), as of 2019, approximately 466 million people worldwide suffered from disabling hearing loss, exceeding 5% of the world's total population (http: / / www.who.int / en / ), severely impacting population quality. While deafness is not fatal, it hinders communication, leading to speech and intellectual developmental disorders, decline in learning and social interaction abilities, reduced work capacity and quality of life, and a range of psychological problems, placing a heavy psychological and economic burden on families and society. Therefore, deafness has become an increasingly urgent social problem that needs to be addressed.

[0004] Deafness has complex causes, with both genetic and environmental factors contributing to its development. Genetic factors account for approximately 50-60%, and are mostly single-gene inheritance. Hereditary deafness exhibits high clinical and genetic heterogeneity. Based on age of onset, it can be classified as congenital deafness, late-onset deafness, and drug-induced deafness. According to its cause, deafness can be classified as conductive deafness, sensorineural deafness, and mixed deafness. Based on whether it is accompanied by other malformations, it is divided into syndrome-type deafness with other symptoms and non-syndromic deafness with deafness as the sole clinical manifestation. Syndromic deafness accounts for approximately 20-30%, while non-syndromic deafness accounts for approximately 70-80%. Based on the inheritance pattern, deafness is mainly classified as autosomal recessive inheritance, autosomal dominant inheritance, sex-linked inheritance, and mitochondrial maternal inheritance.

[0005] Currently, there is no effective treatment for hereditary deafness. A three-tiered prevention strategy is the fundamental approach to its prevention and treatment. Primary prevention is the primary measure, mainly involving population-wide screening for deafness genes and genetic diagnosis of high-risk groups. Specific measures include preconception guidance and fertility guidance for couples carrying deafness-causing mutations, medication and lifestyle guidance for individuals susceptible to environmental factors leading to deafness, and preventing the birth of deaf children through preimplantation genetic diagnosis. Secondary prevention primarily involves prenatal diagnosis of deafness genes during pregnancy to ensure early detection and intervention. Tertiary prevention refers to early detection, early diagnosis, and early treatment of deaf children. Based on early detection, medical interventions aim to promote the development of speech, intelligence, and cognitive functions in deaf children. Identification of deafness-causing genes and research into their mechanisms are crucial prerequisites for implementing this three-tiered prevention strategy. With the widespread adoption of next-generation sequencing, more and more deafness genes are being discovered; nearly 200 deafness genes have been identified to date, including 124 non-syndromic deafness genes (https: / / hereditaryhearingloss.org / ). Although nearly 200 deafness genes have been discovered, the pathogenic genes for a considerable number of patients with hereditary deafness have not yet been identified, and gene diagnosis cannot yet be performed, indicating that there are still new deafness genes to be identified. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention aims to provide the application of the RGS12 gene in deafness detection. This invention, through animal models and samples from deaf individuals, fully demonstrates that the RGS12 (Regulator of GProtein Signaling 12) gene is a novel autosomal recessive deafness gene, which can serve as a new target for deafness gene diagnosis. The discovery of this site will improve the detection rate in clinical deafness gene screening. Based on the above research results, this invention is thus completed.

[0007] Specifically, the present invention relates to the following technical solutions:

[0008] In a first aspect, the invention provides the use of a substance for detecting the RGS12 gene in the preparation of a deafness detection product.

[0009] The substances used to detect the RGS12 gene include, but are not limited to, substances used to detect the expression level, protein modification, amino acid mutations, and gene mutation status of the gene or its encoded protein.

[0010] Specifically, this invention conducted mutation analysis in mice exhibiting a deafness phenotype and discovered a homozygous missense mutation c.2333C>T (NM_173402.2) in exon 7 of the RGS12 gene. This mutation causes the amino acid at position 778 of RGS12 to change from alanine to valine (p.Ala778Val). Simultaneously, it was demonstrated that the A778V missense mutation is a pathogenic variant. Furthermore, in samples from deaf individuals, it was found that deaf patients carried two RGS12 mutations inherited from their parents (p.Ile966 Leu, p.Asp345Glu). Further research in this invention suggests that the main cause of deafness due to RGS12 gene mutations is likely the impact on hair cell function.

[0011] A second aspect of the present invention provides a deafness detection product, the product comprising a substance for detecting the RGS12 gene.

[0012] The substance used to detect the RGS12 gene is the same as that in the first aspect, and will not be described again here.

[0013] A third aspect of the present invention provides a system for detecting deafness, the system comprising:

[0014] i) An analysis unit, the analysis unit comprising: a detection substance selected from the RGS12 gene in a test sample of a subject;

[0015] ii) An assessment unit comprising: determining the condition of the subject based on the status of the RGS12 gene as determined in i).

[0016] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0017] The above-mentioned technical solutions, based on animal models and samples from deaf individuals, fully demonstrate that the RGS12 gene is a novel autosomal recessive deafness gene, which can serve as a new target for deafness gene diagnosis. The discovery of this site will improve the detection rate in the clinical deafness gene screening process.

[0018] In summary, the above-mentioned technical solutions provide new theoretical basis for revealing the mechanism of deafness and lay the foundation for developing early warning models applicable to deafness diseases, thus having good practical application value. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0020] Figure 1 The sequencing results of the Rgs12 gene in spontaneously mutated deaf mice according to an embodiment of the present invention;

[0021] Figure 2 The following are the bioinformatics analysis results of the RGS12 gene mutation in the embodiments of the present invention; wherein, A proves that the mutation site is highly conserved among all species, and BD are predictions of the mutation as a pathogenic mutation using different software respectively;

[0022] Figure 3 This is the sequencing result of the RGS12 gene mutation in a deaf family according to Embodiment 1 of the present invention;

[0023] Figure 4 The above are the immunofluorescence results of hair cells from Rgs12 gene mutant mice in this embodiment of the invention.

[0024] Figure 5 The results of immunofluorescence (A) and scanning electron microscopy (B) of hair cells from Rgs12 gene knockout mice in this embodiment of the invention are shown. Detailed Implementation

[0025] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] In a typical embodiment of the present invention, the application of a substance for detecting the RGS12 gene is provided in the preparation of a deafness detection product.

[0028] The substances used to detect the RGS12 gene include, but are not limited to, substances used to detect the expression level, protein modification, amino acid mutations, and gene mutation status of the RGS12 gene or its encoded protein.

[0029] Specifically, this invention conducted mutation analysis in mice exhibiting a deafness phenotype and discovered a homozygous missense mutation c.2333C>T (NM_173402.2) in exon 7 of the RGS12 gene. This mutation causes the amino acid at position 778 of RGS12 to change from alanine to valine (p.Ala778Val). Simultaneously, it was demonstrated that the A778V missense mutation is a pathogenic variant. Furthermore, in samples from deaf individuals, it was found that deaf patients carried two RGS12 mutations inherited from their parents (p.Ile966 Leu, p.Asp345Glu). Further research in this invention suggests that the main cause of deafness due to RGS12 gene mutations is likely the impact on hair cell function.

[0030] Furthermore, the substances used to detect the RGS12 gene include, but are not limited to, substances based on mass spectrometry, high-performance liquid chromatography, DNA microarray, sequencing, allele-specific probe hybridization, restriction fragment analysis, oligonucleotide ligation detection, single-strand conformation polymorphism analysis, and allele-specific amplification detection. Those skilled in the art can implement these methods using appropriate techniques based on existing known technologies.

[0031] The products described can exist in any existing, known form, such as primers, probes, nucleic acid membrane strips, (gene or protein) chips, formulations, reagent kits, instruments, detection devices, and equipment. Those skilled in the art can realize the above products without inventive effort based on practical experience; therefore, all of the above products fall within the scope of protection of this application.

[0032] In another specific embodiment of the present invention, a deafness detection product is provided, the product comprising a substance for detecting the RGS12 gene.

[0033] The substance used to detect the RGS12 gene is the same as that in the first aspect, and will not be described again here.

[0034] In summary, by detecting the aforementioned pathogenic genes and their mutations, screening, (aided) diagnosis, monitoring, and predictive assessment of deafness (cause) can be achieved. Therefore, the product described herein possesses the aforementioned functions of screening, (aided) diagnosis, monitoring, and predictive assessment of deafness (cause).

[0035] The products described can exist in any existing, known form, such as primers, probes, nucleic acid membrane strips, (gene or protein) chips, formulations, reagent kits, instruments, detection devices, and equipment. Those skilled in the art can realize the above products without inventive effort based on practical experience; therefore, all of the above products fall within the scope of protection of this application.

[0036] In another specific embodiment of the present invention, a system for detecting deafness is provided, the system comprising:

[0037] i) An analysis unit, the analysis unit comprising: a detection substance selected from the RGS12 gene in a test sample of a subject;

[0038] ii) An assessment unit comprising: determining the condition of the subject based on the status of the RGS12 gene as determined in i).

[0039] The sample to be tested can be a human or a non-human sample. The non-human sample can be a non-human mammal sample, specifically a mouse.

[0040] The detection materials for the relevant genes are the same as those in the first aspect mentioned above, and will not be repeated here.

[0041] The subjects' circumstances include screening, (aid) diagnosis, monitoring, and predictive assessment of hearing loss (cause).

[0042] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0043] Example

[0044] I. RGS12 is a novel candidate gene for non-syndromic deafness. The applicant first discovered numerical and structural abnormalities in the cochlear hair cells of some wild-type mice. Auditory brainstem response testing of the offspring of these mice revealed hearing impairment. Through continuous self-crossing and hybridization, the inheritance pattern was preliminarily determined to be autosomal recessive. Subsequently, whole-exome sequencing was used to analyze mutations in mice exhibiting the deafness phenotype. The results showed a homozygous missense mutation c.2333C>T (NM_173402.2) in exon 7 of the Rgs12 gene (Regulator of G Protein Signaling 12). This mutation causes the amino acid at position 778 to change from alanine to valine (p.Ala778Val). Figure 1 ).

[0045] II. Bioinformatics prediction of the A778V missense mutation as a pathogenic variant. Sanger sequencing confirmed that this mutation co-segregates with the deafness phenotype in mice. Conservation analysis showed that this variant site is highly conserved across all species. Figure 2 A), software such as PolyPhen-2, SIFT, and Mutationtaster predicted this variant to be a pathogenic variant. Figure 2 (BD), while querying the ExAC and 1000genome databases shows that the mutation frequency is 0.

[0046] III. RGS12 gene mutations were also found in samples from deaf patients, and the inheritance pattern was consistent with the aforementioned findings. To further clarify the role of RGS12 in the development of hereditary deafness, the applicant screened high-throughput sequencing data from deaf individuals and initially identified a small family carrying a compound heterozygous mutation in the RGS12 gene. In this family of three, the parents had normal hearing and each carried a different mutation. The patient carried two RGS12 mutations inherited from their parents (p.Ile966Leu, p.Asp345Glu), respectively. (See...) Figure 3 .

[0047] IV. The Rgs12 gene is expressed in cochlear hair cells, and mutations affect the morphology of the stereocilia in mouse hair cells. To verify the preliminary mechanism by which RGS12 gene mutation leads to deafness, we examined the hair cells of newborn mice. Through basilar membrane flaps, we found that the hair cells of the mutant mice exhibited abnormal morphology. Compared with wild-type mice, morphological changes were observed in the outer hair cells of the basal, middle, and apical gyri, with the "V"-shaped structure disappearing. Figure 4 ).

[0048] V. Rgs12 gene knockout affects mouse hair cell morphology. Similar to the results in mutant mice, we found that the hair cells in gene knockout mice also exhibited the same morphological changes, with the "V"-shaped structure disappearing. Scanning electron microscopy further confirmed these findings, fully demonstrating that the mutation is a loss-of-function mutation. Furthermore, the main cause of deafness due to RGS12 gene mutation is likely the impairment of hair cell function. Figure 5 ).

[0049] In summary, based on animal models and human samples of deaf individuals, the applicant has fully demonstrated that the RGS12 gene is a novel autosomal recessive deafness gene that can serve as a new target for deafness gene diagnosis. The discovery of this site will improve the detection rate in clinical deafness gene screening.

[0050] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Detection RGS12 Application of genetic material in the preparation of mouse deafness detection products; in, The detection RGS12 Genetic material includes detection of mouse-derived genes. RGS12 The gene mutation site c.2333C>T was used to detect substances at the murine RGS12 protein mutation site p.Ala778Val; the murine source... RGS12 The gene is the transcript shown in the NCBI reference sequence NM_173402.

2.

2. The application as described in claim 1, characterized in that, The detection RGS12 Genetic substances include those based on mass spectrometry, high-performance liquid chromatography, DNA microarray, sequencing, restriction fragment analysis, oligonucleotide ligation detection, single-strand conformation polymorphism analysis, and allele-specific amplification detection.

3. The application as described in claim 1, characterized in that, The product described is one that has functions for screening, assisting in diagnosis, and monitoring deafness.

4. The application as described in claim 3, characterized in that, The products include primers, probes, nucleic acid membrane strips, chips, reagent kits, and equipment.

5. The application as described in claim 4, characterized in that, The product is a gene chip or protein chip.

6. A deafness detection product, characterized in that, The product includes testing. RGS12 Genetic material; Among them, the detection RGS12 The genetic material is as described in the application of claim 1.

7. A system for detecting deafness, the system comprising: i) An analysis unit, the analysis unit comprising: for determining the presence of [a substance] in the test sample of the subject. RGS12 Gene detection material, wherein the subject is a mouse, and; ii) An evaluation unit, the evaluation unit comprising: according to the determination in i) RGS12 The genetic condition was used to determine the subject's condition; Detection RGS12 The genetic material as described in claim 1; The subjects' information includes screening, assisted diagnosis, and monitoring of hearing loss.

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