CN117683778A - GJB6 gene mutant and application thereof - Google Patents

GJB6 gene mutant and application thereof Download PDF

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Publication number
CN117683778A
CN117683778A CN202311701194.3A CN202311701194A CN117683778A CN 117683778 A CN117683778 A CN 117683778A CN 202311701194 A CN202311701194 A CN 202311701194A CN 117683778 A CN117683778 A CN 117683778A
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gene
gjb6
gjb6 gene
mutant
hearing loss
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汤文学
赵月
许红恩
田永安
张森
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Zhengzhou Sanglin Biotechnology Co ltd
Zhengzhou University
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Zhengzhou Sanglin Biotechnology Co ltd
Zhengzhou University
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Abstract

A GJB6 gene mutant, said GJB6 gene mutant having a c.223c > T mutation compared to a wild type GJB6 gene. The invention provides a new pathogenic variation of autosomal dominant hearing loss pathogenic gene GJB6, which can effectively detect whether a biological sample suffers from deafness by detecting whether the gene pathogenic variation exists in the biological sample, further expands and perfects detection and research of hereditary hearing loss, provides a new detection site for diagnosis or treatment of the disease, and a new detection method and path, and has remarkable social and economic benefits.

Description

GJB6 gene mutant and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a GJB6 gene mutant and application thereof.
Background
Hearing Loss (HL) is the most common disease of sensory dysfunction, and worldwide, congenital deafness is about 1.3/mill. It is estimated that more than 50% of severe and extremely severe hearing loss is associated with genetic factors. Hereditary hearing loss can be classified into syndrome type hearing loss and non-syndrome type hearing loss according to whether other system or organ abnormalities are combined. About 30% are syndrome type hearing losses.
With the development of high-throughput sequencing technology, more and more genetic hearing loss related genes are identified, and a basis is provided for molecular diagnosis of the genetic hearing loss, so that more genetic hearing loss is diagnosed and treated. The molecular mechanism of the occurrence of the hearing loss is determined by a gene detection means, so that prenatal gene diagnosis and intervention measures are further adopted, and the method is an effective means for reducing the occurrence rate of the hearing loss and is one of fundamental ways for preventing and treating the hearing loss.
Disclosure of Invention
Aiming at the situation, in order to overcome the defects of the prior art, the invention provides the GJB6 gene mutant and the application thereof, which can effectively screen the carrier with the pathogenic variation of the autosomal dominant acquired progressive hearing loss and prompt the risk of the autosomal dominant acquired progressive hearing loss, and the detection result can provide scientific basis for early diagnosis and differential diagnosis of the autosomal dominant hearing loss and implantation of a hearing aid and an artificial cochlea.
In order to achieve the above purpose, the technical scheme of the invention is that the GJB6 gene mutant has c.223C > T mutation compared with the wild GJB6 gene.
The invention also provides a nucleic acid, which has c.223C > T mutation compared with a wild type GJB6 gene, the nucleotide sequence of the wild type GJB6 gene is shown as SEQ ID NO.1, the 223 position is C, and the codon of the C is 223-225 position CGG.
SEQ ID NO.1：
ATGGATTGGGGGACGCTGCACACTTTCATCGGGGGTGTCAACAAACACTCCACCAGCATCGGGAAGGTGTGGATCACAGTCATCTTTATTTTCCGAGTCATGATCCTCGTGGTGGCTGCCCAGGAAGTGTGGGGTGACGAGCAAGAGGACTTCGTCTGCAACACACTGCAACCGGGATGCAAAAATGTGTGCTATGACCACTTTTTCCCGGTGTCCCACATCCGGCTGTGGGCCCTCCAGCTGATCTTCGTCTCCACCCCAGCGCTGCTGGTGGCCATGCATGTGGCCTACTACAGGCACGAAACCACTCGCAAGTTCAGGCGAGGAGAGAAGAGGAATGATTTCAAAGACATAGAGGACATTAAAAAGCAGAAGGTTCGGATAGAGGGGTCGCTGTGGTGGACGTACACCAGCAGCATCTTTTTCCGAATCATCTTTGAAGCAGCCTTTATGTATGTGTTTTACTTCCTTTACAATGGGTACCACCTGCCCTGGGTGTTGAAATGTGGGATTGACCCCTGCCCCAACCTTGTTGACTGCTTTATTTCTAGGCCAACAGAGAAGACCGTGTTTACCATTTTTATGATTTCTGCGTCTGTGATTTGCATGCTGCTTAACGTGGCAGAGTTGTGCTACCTGCTGCTGAAAGTGTGTTTTAGGAGATCAAAGAGAGCACAGACGCAAAAAAATCACCCCAATCATGCCCTAAAGGAGAGTAAGCAGAATGAAATGAATGAGCTGATTTCAGATAGTGGTCAAAATGCAATCACAGGTTTCCCAAGCTAA
In a further aspect, the invention also provides a construct comprising the gene mutant or the nucleic acid described above.
A mutant protein of a GJB6 gene, wherein compared with the amino acid sequence of a protein expressed by a wild type GJB6 gene, the mutant site is p.Arg75Trp, the amino acid sequence of the protein expressed by the wild type GJB6 gene is shown as SEQ ID NO.2, and the 75 site is 'R'.
SEQ ID NO.2：
MDWGTLHTFIGGVNKHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGCKNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIEDIKKQKVRIEGSLWWTYTSSIFFRIIFEAAFMYVFYFLYNGYHLPWVLKCGIDPCPNLVDCFISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALKESKQNEMNELISDSGQNAITGFPS
The invention also provides a recombinant cell obtained by transforming a receptor cell with the construct or obtained by mutating the GJB6 gene.
The invention also provides application of the nucleic acid or the protein reagent in preparation of a kit, wherein the reagent is used for diagnosing non-syndrome or syndrome type hearing loss, and comprises antibodies, probes, primers and mass spectrometry detection reagents specific for GJB6 gene mutants or GJB6 gene mutant proteins.
The present invention also provides a medicament for treating a syndrome-type or non-syndrome-type hearing loss, the medicament containing an agent that specifically alters the nucleic acid or the protein described above, wherein the agent is an agent based on a gene editing method including at least one selected from shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR-Cas9, CRISPR-Cpf1, and zinc finger nuclease.
The invention also provides a kit for detecting or screening a biological sample of syndrome-type hearing loss, wherein the kit comprises a reagent suitable for detecting at least one of GJB6 gene mutant or GJB6 gene mutant protein, and the reagent comprises an antibody, a probe, a primer and a mass spectrum detection reagent specific to the GJB6 gene mutant or the GJB6 gene mutant protein.
The invention also provides the use of a biological model carrying at least one of the above-mentioned nucleic acids or the above-mentioned proteins for screening a drug.
The biological model is a cell model or an animal model.
The invention provides a new pathogenic variation of autosomal dominant hearing loss pathogenic gene GJB6, which can effectively detect whether a biological sample suffers from deafness by detecting whether the gene pathogenic variation exists in the biological sample, further expands and perfects detection and research of hereditary hearing loss, provides a new detection site for diagnosis or treatment of the disease, and a new detection method and path, and has remarkable social and economic benefits.
Drawings
FIG. 1 is a family diagram of autosomal dominant postoral progressive hearing loss provided by the present invention.
Fig. 2 is a graph of pure tone audiometry results for a portion of the patient shown in fig. 1 in accordance with the present invention.
FIG. 3 is a diagram of Sanger sequencing peaks at position c.223 of the GJB6 gene representing a member of the patient family shown in FIG. 1 provided by the present invention.
FIG. 4 is a graph of hearing loss of a mouse model of the invention (carrying a Gjb R75W mutation) (graph of the trend of change in ABR threshold for mice of different genotypes at different ages over various frequencies).
Detailed Description
The following detailed description of specific embodiments of the invention refers to the accompanying drawings and the detailed description.
The invention particularly provides a GJB6 mutant gene, nucleic acid, protein and application thereof related to syndrome type hearing loss. The provided mutant genes have c.223c > T mutation compared to the wild type GJB6 gene. The mutant gene provided by the invention is a new pathogenic variation related to hearing loss, and can be used for effectively detecting whether a biological sample is suffering from deafness by detecting whether the pathogenic variation of the gene exists in the biological sample, and the detection and research of hereditary hearing loss are further expanded and perfected, so that a new detection site and a new detection method and a new detection way are provided for diagnosis or treatment of the disease.
The invention discovers a new dominant hereditary deafness family named DFNA-SGX in large-scale deafness gene screening and molecular diagnosis (family chart is shown in figure 1). Wherein, ≡indicates normal female, ≡indicates normal male, ≡ ■ indicates male patient, ≡indicates female patient,? ", to be followed. Patients of this family exhibited postlingual progressive hearing loss, as shown in fig. 2, which shows the number, sex, age and genotype of each individual. Numbering is seen in the family chart of fig. 1, het: heterozygous mutant forms; WT: wild type, wherein wild type individuals IV-23 and IV-24 show mild otitis media when tested for hearing. The circles represent the right ear and the asterisks represent the left ear.
The whole genome linkage analysis and whole exome sequencing are carried out on the family, and the result shows that the pathogenic gene is positioned in a 13q11-13q14.13 region, only GJB6 gene c.223C > T variation is formed in the linkage region, and the GJB6c.223C > T variation is primarily determined to be the pathogenic cause of the family. Sanger sequencing was performed on the screened variants (FIG. 3), which were found to co-segregate with disease phenotypes, thus determining GJB6c.223C > T as the causative agent of the family, with the following experimental data:
1. whole exome sequencing
After informed consent was obtained, 3-5ml of peripheral venous blood of the ancestor of the DFNA-SGX family and its family were collected, and edta was anticoagulated, and genomic DNA was extracted according to the protocol provided by the Jin Maige genomic DNA extraction kit (everstate Jin Maige biotechnology limited). After obtaining the genome DNA, the concentration and purity of the DNA were measured by using a Nanodrop One (America Thermo Scientific company), and after quality control, the DNA was obtained by using an ultrasonic breaker (Biorupter)Pico Sonication System, diagenode) fragments the genomic DNA of the precursor and its family members to obtain a DNA fragment of 250bp in length. Fragmented DNA was quality controlled using Agilent 4200TapeStation System and library construction was performed using MGIEasy universal DNA library preparation kit reference kit instructions, capturing of exon regions was performed using MGIEasy Exome Capture V. Double-ended 150bp sequencing was performed using a Huada MGISEQ-2000RS sequencer. Sequencing data was taken off machine, quality control and linker sequence removal were performed using fastp software, then aligned with the ginseng genome (GRCh 37) using BWA software, and mutation identified using GATK Haplotypecaller. Vcfanno software (version number 0.3.1) was used on the resulting VCF file, and external databases (Clinvar, 1 000genome, gnomAD, exAC, d)bNSFP, etc.), the mutations are screened according to the population frequency, genetic patterns, etc., and the screened mutations are rated for mutation pathogenicity by clinicians and genetic counselor based on the recommendations of the american society of medical genetics and genomics for genetic mutation classification standards and guidelines and ClinGen hearing loss expert group for ACMG mutation interpretation.
2. Whole genome linkage analysis
And respectively extracting the mutation of each sample from the obtained VCF file to form an independent VCF file, and compressing the VCF file into a zip file. Linkage analysis was then performed using PEDMINER on-line software (https:// mcg. Ustc. Edu. Cn/bsc/pepdminer /), analysis mode selection autosomal dominant inheritance.
3. Sanger sequencing validation
The candidate pathogenic variants screened for whole exome sequencing detection were verified in pre-sponsors and their families using polymerase chain reaction (polymerase chain reaction, PCR) and Sanger sequencing. The NCBIPrimer-BLAST software is adopted to carry out primer design on the GJB6c.223C > T locus, the primer sequence is that the forward primer is shown as SEQ ID NO.3, the reverse primer is shown as SEQ ID NO.4, and the primers are provided by synthesis of the division of biological engineering (Shanghai). After primer synthesis, the selected GJB6c.223C > T candidate variation was subjected to PCR reaction using a 2×Taq Master Mix kit (offshore protein technologies Co., ltd.) under the following conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94℃for 30 seconds, annealing at 60℃for 30 seconds, extension at 72℃for 1 minute, the cycle of this step was 35 times; extension was carried out at 72℃for 10 minutes. The PCR reaction system was 25. Mu.L, and the amplified products were subjected to 2.2% agarose gel electrophoresis to determine the fragment size, and then purified using a DNA product purification kit (Shanghai Lai maple Biotechnology Co., ltd.) and the purified PCR products were subjected to Sanger sequencing using a sequencer of Applied Biosystems, as shown in Table 1 below.
AGGGGTCAATCCCACATTTCAA(SEQ ID NO.3)；
CCTTTGCCCACTTTTGTCTGTT(SEQ ID NO.4)。
TABLE 1PCR reaction System
All individuals with the disease are found to carry the heterozygous mutation of GJB6c.223C > T through verification, and all individuals with normal hearing are wild type at the site (figure 3), so the GJB6c.223C > T is determined to be the pathogenic gene and mutation of the family.
4. Mouse model construction and hearing test
Single-stranded guide RNA (sgRNA) aiming at R75W mutation is designed according to GJB6 gene sequence, the sgRNA and Cas9 with activity are detected to be transcribed into RNA in vitro, the sgRNA, cas9 mRNA and the doner DNA fragment containing point mutation sequence are injected into fertilized eggs of mice through microinjection and returned into oviduct of a surrogate mouse, PCR and Sanger sequencing are carried out after birth, positive F0 mice are obtained, F1 and F2 mice are obtained through two rounds of propagation, and the genotyping mice are used for subsequent experiments.
The ABR hearing test was performed on different groups of mice, as shown in fig. 4, which shows ABR at 4 to 32kHz frequency for different mice in the closed-field situation. The results show that the ABR threshold was substantially identical for wild-type mice from 3 weeks to 8 weeks postnatal, and that heterozygous mutant mice (GJB 6 mu/+) were not different from wild-type for 3 weeks postnatal, but that hearing loss progressed significantly with increasing week-of-life, and were already total deaf at 6 weeks of age.
Based on an autosomal dominant inheritance deafness family which is automatically collected, through whole-exon sequencing, whole-genome linkage analysis and Sanger sequencing verification, the invention successfully discovers that only c.223C > T mutation of GJB6 gene in a linkage region is co-separated from a disease phenotype, determines the disease mutation of the family, and finally is verified in the family and normal crowd contrast. The GJB6 mutant gene provided by the invention is a new pathogenic variation of hearing loss, and can be used for screening a carrier with the pathogenic variation of autosomal dominant acquired progressive hearing loss by detecting whether the pathogenic variation of the gene exists in a biological sample, prompting the risk of the occurrence of the acquired progressive hearing loss, further expanding and perfecting the detection and research of the hereditary hearing loss, providing a new detection site for the diagnosis or treatment of the disease, a new detection method and a new detection way, and providing scientific basis for the early diagnosis, differential diagnosis and hearing aid and artificial cochlea implantation of the autosomal dominant hearing loss as a detection result.
It should be noted that the above-mentioned embodiments are merely preferred embodiments of the present invention, and the present invention is not limited thereto, and that any equivalent embodiments which can be modified or altered by persons skilled in the art without departing from the scope of the invention are within the scope of the invention.

Claims (10)

1. A GJB6 gene mutant, characterized in that the GJB6 gene mutant has a c.223c > T mutation compared to the wild type GJB6 gene.
2. A nucleic acid, characterized in that the nucleic acid has a c.223c > T mutation compared with a wild type GJB6 gene, and the nucleotide sequence of the wild type GJB6 gene is shown as SEQ ID No. 1.
3. A construct comprising the gene mutant of claim 1 or the nucleic acid of claim 2.
4. The GJB6 gene mutant protein is characterized in that compared with the amino acid sequence of a protein expressed by a wild type GJB6 gene, the mutant site is p.Arg75Trp, wherein the amino acid sequence of the protein expressed by the wild type GJB6 gene is shown as SEQ ID NO. 2.
5. A recombinant cell obtained by transforming a recipient cell with the construct of claim 3 or expressing the GJB6 gene mutein of claim 4.
6. Use of a nucleic acid according to claim 2 or a protein according to claim 4 in the preparation of a kit for the diagnosis of non-syndrome or syndrome-type hearing loss, said reagents comprising antibodies, probes, primers and mass spectrometry detection reagents specific for a GJB6 gene mutant or a GJB6 gene mutein.
7. A medicament for treating hearing loss of syndrome or non-syndrome type, comprising an agent that specifically alters the nucleic acid of claim 2 or the protein of claim 4, wherein the agent is based on an agent comprising at least one gene editing method selected from shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR-Cas9, CRISPR-Cpf1, and zinc finger nuclease.
8. A kit for detecting or screening a biological sample for a syndrome-type hearing loss, comprising reagents suitable for detecting at least one of a GJB6 gene mutant or a GJB6 gene mutein, said reagents comprising antibodies, probes, primers and mass spectrometry detection reagents specific for the GJB6 gene mutant or the GJB6 gene mutein.
9. Use of a biological model for screening a drug, wherein the biological model carries at least one of the nucleic acid of claim 2 or the protein of claim 4.
10. The use according to claim 9, wherein the biological model is a cellular model or an animal model.
CN202311701194.3A 2023-12-12 2023-12-12 GJB6 gene mutant and application thereof Pending CN117683778A (en)

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