CN110878307B

CN110878307B - Gene mutant and application thereof

Info

Publication number: CN110878307B
Application number: CN201811039145.7A
Authority: CN
Inventors: 高雪; 袁永一; 戴朴; 林琼芬; 管李萍; 张建国; 谌于蓝
Original assignee: Chinese PLA General Hospital; BGI Shenzhen Co Ltd
Current assignee: Chinese PLA General Hospital; BGI Shenzhen Co Ltd
Priority date: 2018-09-06
Filing date: 2018-09-06
Publication date: 2021-10-08
Anticipated expiration: 2038-09-06
Also published as: CN110878307A

Abstract

The invention discloses a nucleic acid and a gene mutant related to non-syndromic hereditary hearing loss and application thereof. The non-syndromic hereditary hearing loss associated nucleic acid has the following mutations compared with the wild-type SH3GLB1 gene: c.413C > T and/or c.646A > G. The nucleic acid of the invention is a novel related pathogenic nucleic acid for non-syndromic hereditary hearing loss, and whether the biological sample suffers from non-syndromic hereditary hearing loss can be effectively detected by detecting whether the nucleic acid exists in the biological sample. The discovery of the nucleic acid or the pathogenic mutation site 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 or treatment of the diseases.

Description

Gene mutant and application thereof

Technical Field

The invention relates to the field of biology, in particular to a gene mutant and application thereof, and more particularly relates to nucleic acid, gene mutation, polypeptide, a biological model, a medicament for treating non-syndromic deafness, a kit for detecting non-syndromic deafness, a construct and a recombinant cell.

Background

Deafness (hearing loss) is a general term for a group of auditory dysfunctional diseases. Deafness is a common and frequently encountered disease worldwide. The disease can be caused by the dysfunction of sound transmission or sound perception of the auditory system, and also can be caused by the pathological changes of auditory nerve or central authorities of all levels in auditory conduction paths. The etiology is very complex and diverse, and deafness can be caused by heredity, infection, trauma, improper drug application, immunological diseases, physiological function degeneration, noise, chemical poisoning, psychological factors and the like, wherein the genetic factors play a dominant role. Investigation has shown that the incidence of congenital deafness in newborn is about 1 ‰, and most of them is related to genetic factors, which is called hereditary deafness (HHL), and is caused by chromosomal or gene dysfunction.

The genetic deafness classification method has no unified standard at present, and can be classified into non-syndrome type genetic deafness (NSHHL) and syndrome type deafness (SHHL) according to whether the method is accompanied by the symptoms of other tissues or organs; according to their influence on speech function formation, they can be classified into presbycusis (presngual deafness) and postspeech deafness (postlingual deafness); according to the severity of deafness, it can be classified into mild deafness (mild deafness), moderate deafness (moderate deafness), severe deafness (severe deafness), and very severe deafness (severe deafness); according to the hearing loss of different frequencies, the hearing loss can be divided into low-frequency hearing loss, medium-frequency hearing loss, high-frequency hearing loss, full-frequency hearing loss, and the like.

Non-syndromic deafness refers to the unique symptom of deafness in the affected individuals, has no other genetic damage sexual organ dysfunction, and accounts for about 70% of the genetic deafness. According to the different genetic methods, it is generally classified into autosomal Dominant (DFNA), autosomal recessive (DFNB), sex-linked (sex-linked) and mitochondrial inherited (mitogenic inherited) deafness.

In the classification of non-syndromic deafness, autosomal recessive hereditary deafness accounts for about 75-85%, wherein about 50% of patients worldwide are pathogenic due to GJB2 gene mutation and belong to DFNB1 subtype; the deafness of the remaining 50% of patients is caused by other gene variations, and most of the genes can only explain the onset of 1-2 deafness families. Parents with autosomal recessive hereditary deafness have a 25% probability of producing a child with deafness, a 50% probability of producing a child carrying a pathogenic site of deafness, and a 25% probability of producing a normal child each time they give birth. Thus, the probability that a normal individual in a deaf family is a carrier is 2/3. When a mutation causing DFNB1 is present in a family member, it can be determined by a carrier test whether the family member is a carrier, and the fetus can be detected by prenatal diagnosis.

With the improvement of research level of research technology, molecular genetics and molecular biology, the gene identification work of deafness has advanced greatly, and by 4 months in 2018, 70 ARNSHL (Autosomal Recessed Heart disease) genes are successfully cloned or identified by people. These genes are involved in a wide variety of types, including ion balance-related molecules (components of ion channels and gap junctions), hair cell cytoskeleton components and related molecules (cytoskeletal proteins and molecules), extracellular matrix components and related molecules (extracellular matrix components), transcription factors and activators (transcription factors and activators), and other unclassified gene expression products.

With the development of sequencing technology, more and more genes related to genetic deafness are identified, which provides a basis for the molecular diagnosis of genetic deafness and enables more patients with genetic deafness to be diagnosed and treated. However, due to the strong genetic heterogeneity of hereditary hearing loss, a large number of pathogenic genes are still unidentified, so that the research on the aspect has a large space, and the research on the aspect of enhancing the gene identification is still needed.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art.

The inventor uses the whole exon sequencing technology to sequence 4 samples (I:1, I:2, II:2 and II:3) in an autosomal recessive non-integrated hearing loss (ARNSHL) family to obtain variation data of the samples. The information of family genetic pattern, mutation frequency database, gene expression database and the like is combined to prove that the SH3GLB1 gene can cause autosomal recessive deafness. The inventor also found that: the compound heterozygous mutation of SH3GLB1 c.413C > T p.Thr138Met (allele of mother) and c.646A > G p.Lys216Glu (allele of father) is a mutation causing autosomal recessive nonsynthesis-type deafness (ARNSHL). Furthermore, the inventor discloses two new mutations of SH3GLB1 gene, and proves that the new mutations are the pathogenic genes of autosomal recessive hereditary hearing loss for the first time, enriches the mutation spectrum of the hearing loss gene, and provides a genetic basis for developing the molecular diagnosis of hereditary hearing loss.

In a first aspect of the invention, the invention features a nucleic acid. According to an embodiment of the invention, the nucleic acid has the following mutations compared to the wild-type SH3GLB1 gene: c.413C > T and/or c.646A > G. The inventor firstly discovers that SH3GLB1 gene is related to deafness, and finds that c.413C > T and/or c.646A > G mutation of SH3GLB1 gene is closely related to pathogenesis of non-syndrome type deafness (such as non-syndrome type autosomal recessive deafness), so that whether a biological sample suffers from non-syndrome type hereditary deafness (such as non-syndrome type autosomal recessive hereditary deafness) can be effectively detected by detecting whether the nucleic acid exists in the biological sample.

In a second aspect of the invention, the invention features a genetic mutation. According to the examples of the present invention, there are c.413c > T and/or c.646a > G mutations compared to the wild-type SH3GLB1 gene. The inventor firstly discovers that SH3GLB1 gene is related to deafness, and finds that c.413C > T and/or c.646A > G mutation of SH3GLB1 gene is closely related to pathogenesis of non-syndrome type hereditary deafness (such as non-syndrome type autosomal recessive hereditary deafness), so that whether a biological sample suffers from non-syndrome type hereditary deafness (such as non-syndrome type autosomal recessive hereditary deafness) can be effectively detected by detecting whether the gene mutation occurs in the biological sample.

In a third aspect of the invention, the invention features a polypeptide. According to an embodiment of the present invention, the amino acid sequence of the polypeptide expressed by the wild-type SH3GLB1 gene has the following mutations compared to the amino acid sequence of the polypeptide: p.Thr138Met and/or p.Lys216Glu. As described above, the c.413C.sub.T and/or c.646A.sub.G mutation of SH3GLB1 gene is closely related to the onset of non-syndromic hereditary hearing loss (e.g., non-syndromic autosomal dominant deafness), and thus, it can be said that the protein expressed by the c.413C.sub.T and/or c.646A.sub.G mutant SH3GLB1 gene, a polypeptide having a mutation in p.ThrMet 138and/or p.Lys216Glu, is closely related to the onset of non-syndromic hereditary hearing loss (e.g., non-syndromic autosomal recessive hearing loss), and further, by detecting the presence or absence of the above polypeptide in a biological sample, it is possible to effectively detect whether or not a biological sample suffers from non-syndromic hereditary hearing loss (e.g., non-syndromic autosomal recessive hereditary hearing loss).

In a fourth aspect, the invention provides the use of a reagent for detecting a nucleic acid as hereinbefore described or a genetic mutation as hereinbefore described or a polypeptide as hereinbefore described in the manufacture of a kit or device. According to an embodiment of the invention, the kit or device is for diagnosing non-syndromic genetic deafness. As mentioned above, the nucleic acid, gene mutation, polypeptide described above are closely related to the onset of non-syndromic hereditary hearing loss, and further the reagent for detecting the nucleic acid described above or the gene mutation described above or the polypeptide described above can be used to prepare a kit or device, and the obtained kit or device can effectively screen out biological samples suffering from non-syndromic hereditary hearing loss, especially non-syndromic autosomal recessive hereditary hearing loss.

In a fifth aspect of the invention, the invention proposes the use of a biological model for screening a drug. According to an embodiment of the invention, the biological model carries at least one of the following: (1) the nucleic acid of claim 1; (2) a mutation of the gene of claim 2; (3) expressing the polypeptide of claim 3. It is to be noted that "the biological model carries the aforementioned nucleic acid" means that the biological model of the present invention carries a nucleic acid sequence of an SH3GLB1 gene mutant having c.413c > T and/or c.646a > G mutation as compared with the wild-type SH3GLB1 gene; "biological models carry the aforementioned gene mutation" means that the biological models of the present invention carry SH3GLB1 gene having a mutation of c.413C > T and/or c.646A > G as compared with the wild-type SH3GLB1 gene. By "biological model carries the aforementioned polypeptide" is meant that the biological model of the invention carries a polypeptide having a p.thr138met and/or p.lys216glu mutation compared to the wild-type SH3GLB1 gene. The biological model according to the embodiment of the present invention can be effectively used as a model for the related research of non-syndromic hereditary hearing loss, especially non-syndromic autosomal recessive hereditary hearing loss.

In a sixth aspect, the present invention proposes the use of an agent which specifically alters a nucleic acid as described above or a mutation in a gene as described above, in the manufacture of a medicament for the treatment of non-syndromic genetic deafness. It is to be noted that the specific alteration is such that the mutated nucleic acid or mutated site of the gene is restored to its original wild-type state or other non-pathogenic state without substantially affecting other sequences in the genome of the individual. As described above, the aforementioned nucleic acids or the aforementioned gene mutations are closely related to the onset of non-syndromic hereditary hearing loss, particularly non-syndromic autosomal recessive hereditary hearing loss, and thus, a drug prepared from an agent that specifically alters the aforementioned nucleic acids or the aforementioned gene mutations can be effectively used for treating non-syndromic hereditary hearing loss, particularly non-syndromic autosomal recessive hereditary hearing loss.

In a seventh aspect of the invention, the invention proposes a medicament for the treatment of non-syndromic hereditary hearing loss. According to an embodiment of the invention, the medicament comprises: an agent which specifically alters the aforementioned nucleic acid or the aforementioned gene mutation. It is to be noted that the specific alteration is such that the mutated nucleic acid or mutated site of the gene is restored to its original wild-type state or other non-pathogenic state without substantially affecting other sequences in the genome of the individual. As described above, the aforementioned nucleic acids or the aforementioned gene mutations are closely related to the onset of non-syndromic hereditary hearing loss, particularly non-syndromic autosomal recessive hereditary hearing loss, and thus, a drug comprising an agent that specifically alters the aforementioned nucleic acids or the aforementioned gene mutations can be effectively used for the treatment of non-syndromic hereditary hearing loss, particularly non-syndromic autosomal recessive hereditary hearing loss.

In an eighth aspect of the invention, a method of screening a biological sample for non-syndromic hereditary hearing loss is provided. According to an embodiment of the invention, the method comprises the steps of: extracting a nucleic acid sample from a biological sample; determining the nucleic acid sequence of the nucleic acid sample; and (3) judging whether the biological sample has non-syndrome genetic deafness or not by judging whether the nucleic acid sequence of the nucleic acid sample or the complementary sequence thereof has c.413C > T and c.646A > G mutations compared with the wild-type SH3GLB1 gene, wherein the 413 and 646 sites of the nucleic acid sequence of the nucleic acid sample or the complementary sequence thereof have c.413C > T and c.646A > G mutations compared with the wild-type SH3GLB1 gene and are indicative of the biological sample having non-syndrome genetic deafness. According to the embodiment of the invention, the fact that the 413 and 646 sites of the gene have only c.413C > T and c.646A > G mutations compared with the wild-type SH3GLB1 gene means that the 413 site base of the allele of SH3GLB1 on the chromosome of an organism is mutated from C to T and the 646 site base is mutated from A to G compared with the wild-type SH3GLB1 gene. By the method for screening the biological sample suffering from non-syndrome type hereditary hearing loss, disclosed by the embodiment of the invention, the biological sample suffering from non-syndrome type hereditary hearing loss, particularly non-syndrome type autosomal recessive hereditary hearing loss can be effectively screened.

In a ninth aspect of the invention, the invention provides a construct. According to an embodiment of the invention, the construct comprises the nucleic acid or the genetic mutation as described above. It is to be noted that the expression "the construct comprises a nucleic acid as described above" means that the construct of the invention comprises a nucleic acid having a c.413c > T and/or c.646a > G mutation compared to the wild-type SH3GLB1 gene. By "the construct comprises a gene mutation as described above" is meant that the construct of the invention comprises an SH3GLB1 gene having a c.413c > T and/or c.646a > G mutation compared to the wild-type SH3GLB1 gene. Thus, the recombinant cells obtained by transforming the receptor cells with the constructs according to the embodiments of the present invention can be effectively used as a model for research related to non-syndromic hereditary hearing loss, particularly non-syndromic autosomal recessive hearing loss.

In a tenth aspect of the invention, a recombinant cell is provided. According to an embodiment of the invention, said recombinant cell is obtained by transforming a recipient cell with a construct as described above or expressing a polypeptide as described above. According to some embodiments of the invention, the recombinant cells of the invention can be effectively used as a model for research related to non-syndromic hereditary hearing loss, in particular to non-syndromic autosomal recessive hereditary hearing loss.

In an eleventh aspect of the invention, a kit for detecting non-syndromic hereditary hearing loss is provided. According to an embodiment of the invention, the kit comprises reagents for detecting the nucleic acids described above, and/or reagents for detecting the genetic mutations described above, and/or reagents for detecting the polypeptides described above. As described above, the nucleic acids, gene mutations and polypeptides described above are closely related to the onset of non-syndromic hereditary hearing loss, and thus can be used in a kit comprising reagents effective for detecting the nucleic acids described above or the gene mutations described above or the polypeptides described above, to effectively screen a biological sample suffering from non-syndromic hereditary hearing loss, particularly non-syndromic autosomal recessive hereditary hearing loss.

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

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a family map according to an embodiment of the present invention;

FIG. 2 is a pure tone audiometric chart of a patient according to an embodiment of the present invention, wherein the horizontal axis represents the frequency of sound (i.e., sound tone in Hz), the left-to-right axis represents the low-to-high sound tone, the vertical axis represents the intensity of sound (i.e., sound level in dB), the top-to-bottom axis represents the small-to-large sound, II:2 corresponds to severe sensorineural deafness (binaural), II:3 corresponds to moderate sensorineural deafness (left), and severe sensorineural deafness (right);

FIG. 3 is a graph of SH3GLB1 c.413C > T (p.Thr138Met) and c.646A > G (p.Lys216Glu) Sanger-verified peaks according to an example of the present invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

It is to be understood by those skilled in the art that the position of the wild-type SH3GLB1 gene sequence used in the present invention is based on the sequence of the wild-type SH3GLB1 gene in the human genome, but when the wild-type SH3GLB1 gene exists in other species, the sequence may be different, and the corresponding position in the wild-type SH3GLB1 gene of the species can be obtained by aligning the wild-type SH3GLB1 gene of the species with the wild-type SH3GLB1 gene of human.

It should be noted that the non-syndromic autosomal recessive deafness is an autosomal recessive disease, which means that SH3GLB1 gene on the autosomal chromosome of a biological individual needs c.413c > T and c.646a > G mutations to occur at the corresponding sites of alleles at the same time to cause disease, for example, c.413c > T mutation in the allele of SH3GLB1 gene is denoted as a, c.413c > T mutation does not occur as a, c.646a > G mutation in the allele of SH3GLB1 gene is denoted as B, c.413c > T mutation does not occur as B, and if the genotype is aabb, the biological individual suffers from non-syndromic autosomal recessive deafness. Thus, in the method of the invention for screening a biological sample for non-syndromic genetic deafness, reference to "mutations at positions 413 and 646 thereof, and only c.413c > T and c.646a > G as compared to the wild-type SH3GLB1 gene" means that both alleles of SH3GLB1 have mutations at positions 413 and 646 thereof, and only c.413c > T and c.646a > G.

Nucleic acids

For the purposes of the present description and claims, reference to nucleic acids will be understood by those skilled in the art to include virtually either or both of the complementary strands. For convenience, in the present specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. For example, reference to the sequence of the SH3GLB1 gene actually includes the complementary sequence thereof. One skilled in the art will also appreciate that one strand may be used to detect the other strand and vice versa.

The SH3GLB1 gene mutant has c.413C > T and/or c.646A > G mutation compared with wild SH3GLB1 gene. The inventor firstly proposes the SH3GLB1 gene as a pathogenic gene of non-syndromic autosomal recessive hereditary hearing loss, and firstly discovers that c.413C > T and c.646A > G mutations of the SH3GLB1 gene can cause patients to suffer from the pathogenic gene of non-syndromic autosomal recessive hereditary hearing loss.

It should be noted that the cDNA sequence of SH3GLB1 wild-type gene can be obtained from the following website:

http://grch37.ensembl.org/Homo_sapiens/Transcript/Summarydb＝core；g ＝ENSG00000097033；r＝1:87170259-87213867；t＝ENST00000370558。

the c.413C > T and/or c.646A > G mutation is positioned by a cDNA sequence corresponding to the website.

Gene mutation

Polypeptides

According to an embodiment of the invention, the nucleotide sequence of the polypeptide is compared with the amino acid sequence of the polypeptide expressed by the wild-type SH3GLB1 geneThe amino acid sequence of the polypeptide expressed by the wild-type SH3GLB1 gene has the following acquisition site with the mutations of p.Thr138Met and p.Lys216Glu:http://grch37.ensembl.org/Homo_sapiens/ Transcript/Summarydb＝core；g＝ENSG00000097033；r＝1:87170259-87213867；t＝ ENST00000370558。the p.Thr138Met and p.Lys216Glu mutation is positioned by the amino acid sequence of the polypeptide corresponding to the website.

Use of reagent for detecting nucleic acid, gene mutation and polypeptide in preparation of kit or equipment

In a fourth aspect, the invention provides the use of a reagent for a nucleic acid as hereinbefore described or a genetic mutation as hereinbefore described or a polypeptide as hereinbefore described in the manufacture of a kit or device. According to an embodiment of the invention, the kit or device is for diagnosing non-syndromic genetic deafness. As mentioned above, the nucleic acid, the gene mutation, the polypeptide are closely related to the onset of non-syndromic hereditary hearing loss, and then the reagent for detecting the nucleic acid, the gene mutation or the polypeptide is used for preparing a kit or a device, and the obtained kit or device can effectively screen out biological samples suffering from non-syndromic hereditary hearing loss, especially non-syndromic autosomal recessive hereditary hearing loss.

According to an embodiment of the invention, the non-syndromic hereditary hearing loss is non-syndromic autosomal recessive hereditary hearing loss.

According to an embodiment of the invention, the reagent comprises at least one of an antibody specific for at least one of the nucleic acid, the genetic mutation and the polypeptide, a probe, a primer and a mass spectrometric detection reagent. For example, the inventors can detect the presence of the above mutation in a test sample by the specific binding of an antibody specifically recognizing the polypeptide to the polypeptide, i.e., the presence of the above polypeptide is detected by the interaction of a specific antibody with an antigen; the inventors can also identify the presence of the nucleic acid or gene mutation by designing in advance a probe that specifically recognizes the nucleic acid or gene mutation and complementarily pairing the probe with a nucleic acid fragment in which the site of the nucleic acid or gene mutation is located; the inventors can also design specific primers for amplifying the exons in which the gene mutations are located, and then determine whether the gene mutations exist through gene amplification and sequencing; the inventors also determined the presence of the p.Thr138Met and/or p.Lys216Glu mutated polypeptide by mass spectrometry to detect m/z of the polypeptide. According to the specific embodiment of the invention, at least one of the antibody, the probe, the primer and the mass spectrometric detection reagent can specifically and highly sensitively screen out the nucleic acid or the gene mutation or the polypeptide, and further specifically and highly sensitively screen out a biological sample suffering from non-syndrome type hereditary hearing loss, especially suffering from non-syndrome type autosomal recessive hereditary hearing loss, and further can be effectively used for preparing a kit or a device for screening the biological sample suffering from non-syndrome type hereditary hearing loss, especially suffering from non-syndrome type autosomal recessive hereditary hearing loss.

Biological model

In a fifth aspect of the invention, the invention proposes the use of a biological model for screening a drug. According to an embodiment of the invention, the biological model carries at least one of the following: (1) the nucleic acid of claim 1; (2) a mutation of the gene of claim 2; (3) expressing the polypeptide of claim 3. It is to be noted that "the biological model carries the aforementioned nucleic acid" means that the biological model of the present invention carries a nucleic acid sequence of an SH3GLB1 gene mutant having c.413c > T and c.646a > G mutations compared with the wild-type SH3GLB1 gene; "biological models carry the aforementioned gene mutations" means that the biological models of the present invention carry SH3GLB1 gene having mutations of c.413C > T and c.646A > G compared to the wild-type SH3GLB1 gene. "biological model carries the aforementioned polypeptide" means that the biological model of the present invention carries a polypeptide having p.thr138met and p.lys216glu compared to the wild-type SH3GLB1 gene. The biological model according to the embodiment of the present invention can be effectively used as a model for the related research of non-syndromic hereditary hearing loss, especially non-syndromic autosomal recessive hereditary hearing loss.

According to an embodiment of the invention, the biological model is a cellular model or an animal model.

Use of agents for the manufacture of a medicament

According to embodiments of the invention, the agent is an agent based on at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR-Cas9, CRISPR-Cpf1, and zinc finger nuclease. For example, the CRISPR-Cas9 realizes genome modification mainly through three ways of gene knockout, introduction of special variation and site-directed transgene, based on the method of CRISPR-Cas9, the inventors can design sgRNA and synthesize gRNA of the sequence, co-express the gRNA and d Cas9 in cells, and mediate d Cas9 protein to bind with a target DNA region through the gRNA, thereby realizing repair or change of a specific site.

Medicine for treating non-syndromic hereditary deafness

Method for screening biological samples for non-syndromic hereditary hearing loss

First, a nucleic acid sample is extracted from a biological sample. According to the embodiment of the present invention, the type of the biological sample is not particularly limited as long as a nucleic acid sample reflecting the presence or absence of a mutation in the SH3GLB1 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. Therefore, the sampling and detection can be conveniently carried out, and the efficiency of screening the biological sample suffering from the non-syndromic hereditary hearing loss can be further improved. The term "nucleic acid sample" as used herein is to be understood in a broad sense according to the embodiments of the present invention, and may be any sample capable of reflecting the presence or absence of a mutation in the SH3GLB1 gene in a biological sample, such as whole genomic DNA extracted directly from the biological sample, or a portion of the whole genome containing the coding sequence of the SH3GLB1 gene, such as total RNA extracted from the biological sample, or mRNA extracted from the biological sample. According to one embodiment of the invention, the nucleic acid sample is whole genomic DNA. Therefore, the source range of the biological sample can be expanded, and a plurality of information of the biological sample can be determined simultaneously, so that the efficiency of screening the biological sample suffering from non-syndromic genetic deafness can be improved. In addition, according to an embodiment of the present invention, for using RNA as the nucleic acid sample, extracting the nucleic acid sample from the biological sample may further include: extracting an RNA sample from the biological sample, preferably the RNA sample is mRNA; and obtaining a cDNA sample by reverse transcription reaction based on the obtained RNA sample, the obtained cDNA sample constituting a nucleic acid sample. Therefore, the efficiency of screening biological samples suffering from non-syndromic hereditary hearing loss by using RNA as a nucleic acid sample can be further improved.

Next, after obtaining the nucleic acid sample, the nucleic acid sample may be analyzed, thereby enabling determination of the nucleic acid sequence of the obtained nucleic acid sample. According to embodiments of the present invention, the method and apparatus for determining the nucleic acid sequence of the resulting nucleic acid sample are not particularly limited. According to embodiments of the present invention, the nucleic acid sequence of a nucleic acid sample may be determined by a sequencing method. Methods and apparatuses that may be used to perform sequencing according to embodiments of the present invention are not particularly limited. According to embodiments of the present invention, second generation sequencing techniques may be employed, as well as third generation and fourth generation or more advanced sequencing techniques. According to embodiments of the invention, a nucleic acid sequence may be sequenced using at least one of BGISEQ-500, BGISEQ-500RS, HISEQ2000, SOLID, 454, and a single molecule sequencing device. Therefore, by combining the latest sequencing technology, the higher sequencing depth can be achieved for a single site, and the detection sensitivity and accuracy are greatly improved, so that the characteristics of high throughput and deep sequencing of the sequencing devices can be utilized to further improve the efficiency of detecting and analyzing the nucleic acid sample. Therefore, the accuracy and the precision of the subsequent analysis of the sequencing data can be improved. Thus, according to embodiments of the present invention, determining the nucleic acid sequence of the nucleic acid sample may further comprise: firstly, aiming at the obtained nucleic acid sample, constructing a nucleic acid sequencing library; and sequencing the obtained nucleic acid sequencing library so as to obtain a sequencing result consisting of a plurality of sequencing data. According to some embodiments of the invention, the resulting nucleic acid sequencing library may be sequenced using at least one selected from the group consisting of BGISEQ-500, BGISEQ-500RS, HISEQ2000, SOLID, 454, and single molecule sequencing devices. In addition, according to the embodiment of the invention, the nucleic acid sample can be screened to enrich SH3GLB1 gene exon, and the screening enrichment can be performed before constructing a sequencing library, during constructing the sequencing library, or after constructing the sequencing library. Exon-targeted sequence enrichment systems such as: the capture chip of the Huada major autonomous exon, and other exons or target region capture platforms such as active SureSelect, Nimblegen and the like enrich target fragments. According to one embodiment of the present invention, constructing a nucleic acid sequencing library for a nucleic acid sample further comprises: performing PCR amplification on a nucleic acid sample by using at least one primer selected from SH3GLB1 gene specific primers; and constructing a nucleic acid sequencing library aiming at the obtained amplification products. Therefore, SH3GLB1 gene exon can be enriched through PCR amplification, so that the efficiency of screening biological samples suffering from non-syndromic hereditary hearing loss can be further improved. According to the present example, the sequence of primers specific to SH3GLB1 gene is not particularly limited, and can be obtained by, for example, on-line design using Primer3.0 with reference to the human genome sequence database GRCh37.1/hg19, such as UCSC (http:// genome. UCSC. edu /), primers of candidate genes can be designed and synthesized using Primer3(version 0.4.0, http:// Primer3.ut. ee /) and Primer specificity can be verified using Primer-BLAST (http:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST /). According to some specific examples of the invention, the SH3GLB1 gene specific primer has the sequence as shown in SEQ ID NO: 1-2; aiming at c.646A > G mutation, the SH3GLB1 gene specific primer has the nucleotide sequence shown in SEQ ID NO: 3-4. The inventors have surprisingly found that by using SEQ ID NO: 1-4 (as shown in table 2 below), the amplification of the whole genome sequence of the SH3GLB1 gene mutation can be accomplished significantly and effectively in the PCR reaction system. It is noted that, SEQ ID NO: 1-4 are unexpectedly obtained by the present inventors after a hard work.

Table 2:

with regard to the methods and procedures for constructing sequencing libraries for nucleic acid samples, those skilled in the art may make appropriate selections based on different sequencing techniques, and with regard to the details of the procedures, see the manufacturers of sequencing instruments such as the protocols provided by Illumina, see, for example, the Multiplexing Sample Preparation Guide (Part # 1005361; Feb 2010) or Paired-End Sample Preparation Guide (Part # 1005063; Feb 2010), incorporated herein by reference. The method and apparatus for extracting a nucleic acid sample from a biological sample according to an embodiment of the present invention are not particularly limited, and may be performed using a commercially available nucleic acid extraction kit.

It should be noted that the term "nucleic acid sequence" used herein is to be understood in a broad sense, and may be complete nucleic acid sequence information obtained by assembling sequencing data obtained by sequencing a nucleic acid sample, or may be nucleic acid sequences directly obtained by using sequencing data (reads) obtained by sequencing a nucleic acid sample, as long as the nucleic acid sequences contain coding sequences corresponding to SH3GLB1 gene.

Finally, after determining the nucleic acid sequence of the nucleic acid sample, the reference sequences corresponding to the nucleic acid sequences of the obtained nucleic acid sample are aligned, and when the obtained nucleic acid sequence has the aforementioned mutation, the biological sample is indicated to suffer from non-syndromic genetic deafness. Therefore, by the method for screening the biological sample suffering from non-syndromic genetic deafness according to the embodiment of the invention, the biological sample suffering from non-syndromic genetic deafness can be effectively screened. The method and apparatus for aligning a nucleic acid sequence with a corresponding wild-type gene sequence according to embodiments of the present invention are not particularly limited and may be performed using any conventional software, and according to embodiments of the present invention, alignment may be performed using SOAPALIGNER/SOAP 2.

It should be noted that the use of the "method for screening a biological sample suffering from non-syndromic genetic deafness" according to the embodiment of the present invention is not particularly limited, and for example, it can be used as a screening method for non-diagnostic purposes.

Construct and recombinant cell

In a ninth aspect of the invention, the invention provides a construct. According to an embodiment of the invention, the construct comprises the nucleic acid or the genetic mutation as described above. It is to be noted that the phrase "the construct comprises the nucleic acid as described above" means that the construct of the present invention comprises the nucleic acid having the c.413c > T and c.646a > G mutations compared to the wild-type SH3GLB1 gene. By "the construct comprises a gene mutation as described above" is meant that the construct of the invention comprises an SH3GLB1 gene having a c.413c > T and/or c.646a > G mutation compared to the wild-type SH3GLB1 gene. Thus, the recombinant cells obtained by transforming the receptor cells with the constructs according to the embodiments of the present invention can be effectively used as a model for research related to non-syndromic hereditary hearing loss, particularly non-syndromic autosomal recessive hearing loss. The type of the recipient cell is not particularly limited, and may be, for example, an escherichia coli cell or a mammalian cell, and the recipient cell is preferably derived from a mammal.

The term "construct" as used in the present invention refers to a genetic vector comprising a specific nucleic acid sequence and capable of transferring the nucleic acid sequence of interest into a host cell to obtain a recombinant cell. According to an embodiment 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, and is preferably a plasmid. The plasmid is used as a genetic carrier, has the characteristics of simple operation, capability of carrying larger fragments and convenience for operation and treatment. The form of the plasmid is not particularly limited, and may be a circular plasmid or a linear plasmid, and may be either single-stranded or double-stranded. The skilled person can select as desired. The term "nucleic acid" used in the present invention may be any polymer containing deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, the length of which is not subject to any particular limitation. For constructs used to construct recombinant cells, it is preferred that the nucleic acid be DNA, as DNA is more stable and easier to manipulate than RNA.

According to the embodiment of the present invention, the kind of the recipient cell is not particularly limited, and may be, for example, an escherichia coli cell, a mammalian cell, and preferably, the recipient cell is derived from a non-human mammal.

Kit for detecting non-syndromic hereditary hearing loss

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Unless otherwise indicated, the techniques used in the examples are conventional and well known to those skilled in the art, and may be performed according to the third edition of the molecular cloning, laboratory Manual, or related products, and the reagents and products used are also commercially available. Various procedures and methods not described in detail are conventional methods well known in the art, and the sources, trade names, and components of the reagents used are indicated at the time of first appearance, and the same reagents used thereafter are the same as those indicated at the first appearance, unless otherwise specified.

Examples

The inventor screens candidate variation of a nonsynthetic autosomal recessive deafness family by exon sequencing, screens out nonpathogenic nucleotide variation by bioinformatics analysis, reduces the number of the candidate variation, designs a primer for the candidate variation, carries out PCR amplification, product purification and Sanger sequencing, carries out candidate variation detection on members in the family and normal control people, and defines the relationship between gene mutation and disease by coseparation analysis. Compared with homologous genes in other organisms, biological software predicts the pathogenicity of mutation, finds the relation between SH3GLB1 gene and auditory system through a mouse model, and analyzes the expression condition of polypeptide. The method comprises the following specific steps:

1. collecting samples:

the inventor collects a Chinese Han nationality genetic deafness family (the family is shown in figure 1), the family is composed of 5 members, 2 patients are diagnosed, 2 existing patients are 2, 2 patients (II:2, II:3) are tested by pure tone audiometry (the pure tone audiometry is shown in figure 2); 2 samples (I:1, I:2) of human blood with normal phenotype were collected. 2ml of peripheral blood sample is collected from each sample, EDTA is added for anticoagulation, and the samples are preserved at-80 ℃.

2. DNA extraction

Extracting DNA from a peripheral Blood sample by adopting an OMEGA Blood DNA Midi Kit whole Blood DNA extraction Kit, wherein the extraction steps are as follows:

(1) taking 2mL of whole blood sample, adding 150 uL of OB Protease, 2.1mL of Buffer BL and 20 uL of RNase A, whirling at the maximum speed for 1 minute, and thoroughly mixing;

(2) carrying out water bath at 65 ℃ for 15-20 minutes, and whirling for 5 times in the water bath process;

(3) adding 2.2mL of absolute ethyl alcohol, whirling at the maximum speed for 30 seconds, and thoroughly mixing;

(4) transferring 3.5mL of lysate into a 15mL centrifuge tube with a filter column, centrifuging for 5 minutes at 4000 rpm, taking out the filter column, pouring out the filtered liquid, and putting back for filtration;

(5) adding the remaining lysate in the step 3 into a 15mL centrifuge tube with a filter column, centrifuging for 5 minutes at 4000 rpm, taking out the filter column, pouring out the filtered liquid, and putting back to the filter column;

(6) adding 3mL of HB Buffer, washing the filter column, centrifuging for 5 minutes at 4000 rpm, taking out the filter column, pouring out the filtered liquid, and putting back to the filter column;

(7) adding 3mL of DNA Wash Buffer, centrifuging for 5 minutes at 4000 rpm, taking out the filter column, pouring out the filter liquid, and putting back to the filter column;

(8) adding 3mL of DNA Wash Buffer again, centrifuging for 5 minutes at 4000 rpm, taking out the filter column, pouring out the filter liquid, and putting back to the filter column;

(9) centrifuging for 15 minutes at 4000 revolutions, and drying the filter column;

(10) transferring the filter column to a new 15mL centrifuge tube, adding 500uL of 70 ℃ Elution Buffer, standing for 5 minutes at room temperature, centrifuging for 5 minutes at 4000 rpm, and collecting a filtrate containing DNA;

(11) the filter column was again transferred to a new 15mL centrifuge tube, 500uL of 70 ℃ Elution Buffer was added, the mixture was allowed to stand at room temperature for 5 minutes, centrifuged at 4000 rpm for 5 minutes, and the filtrate containing DNA was collected.

3. Exon capture sequencing and variation analysis

The inventors performed whole exon sequencing and data analysis on 4 samples (I:1, I:2, II:3) in the pedigree. Based on an Illumina Hiseq2000 high-throughput sequencing platform, a NimbleGen SeqCap EZ Exome Library V3.0 chip is adopted to carry out whole exon capture sequencing on the 4 samples, and the method specifically comprises the following steps:

(1) sample preparation

Taking the genome DNA of the peripheral blood samples of the 4 samples (I:1, I:2, II:2 and II:3), respectively, measuring the concentration and purity of the DNA by using a spectrophotometer and a gel electrophoresis method, wherein the OD260/OD280 of the genome DNA of each sample is between 1.7 and 2.0, the concentration is not less than 200 ng/mu L, and the total amount is not less than 30 mu g for later use.

(2) Library construction and sequencing

Each genomic DNA sample was randomly fragmented into fragments of about 150-200bp by the adaptive high-focus ultrasound technique (Covaris), and then the library was prepared by ligating adaptors at both ends of the fragments according to the manufacturer's instructions (see: http:// www.illumina.com/Illumina/Solexa Standard library construction).

The library preparation and sequencing procedures were as follows:

a) designing a primer: the primers used for library construction were synthesized by Biotech, and the Primer specificity was verified using Primer-BLAST (http:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST /), with the Primer sequences shown in Table 3:

table 3:

B) and (3) amplifying the extracted DNA sample by using the designed primers, wherein a PCR amplification system and amplification conditions are as follows:

and (3) PCR reaction system:

and (3) PCR reaction conditions:

c) analysis of PCR products: subjecting the PCR product to electrophoresis in 1.5% agarose gel (selecting appropriate voltage according to the size of the amplified fragment), and analyzing the electrophoretogram;

d) selecting a sample with single band type and higher concentration, purifying the sample by a PCR product to obtain a sequencing library,

and (4) performing computer sequencing after the library is detected to be qualified so as to obtain original sequencing data. Sequencing is carried out according to an Illumina standard protocol of clustering and sequencing, wherein a sequencing platform is Illumina Genome Analyzer II, the reading length is 90bp, and the average sequencing depth of a sample is 138X.

(3) Mutation detection and screening

The sequencing output data are sequentially subjected to preliminary statistical analysis, SNP detection and annotation and prediction of amino acid substitution, and the method mainly comprises the following steps:

performing basic data analysis statistics on sequencing output data: analyzing the length of the measured sequences, counting the number of reads and the yield of data, comparing the reads sequence with a reference genome sequence, counting the Coverage (Coverage) and the sequencing Depth (Depth) of the target region compared to the genome reads to be referred to, and the like. And obtaining sample basic information captured by the exon according to the statistical result of the basic data, and judging whether the data meets the requirements.

The high quality raw reads of each sample are aligned to a reference Genome (hg19) by using SOAPaligner (v2.21) and Burrows-Wheeler Aligner (BWA, v0.7.10) alignment software, then SNP and Indel are detected by using Genome Analysis Toolkit (GATK, v3.3-0) software, variation annotation is carried out on the detected variation by using Variable Effect Predictor (VEP), and frequency databases such as 1KG/ESP/HapMap/ExAC/gnomaD and information such as OMIM, GO, KEGG and hazard prediction are added. According to the prevalence rate of non-syndromic autosomal recessive hereditary hearing loss (ARNSHL), high frequency (1KG/ESP/HapMap/ExAC >0.005) and variation of intron region were filtered out. Then, according to the phenotype and the family genetic pattern of the patient, namely, a parental heterozygous mutation, a child homozygous mutation or a parental gene with one mutation and two mutations in both children (compound heterozygous), the mutation on the SH3GLB1 gene is found to be qualified, and the two missense mutations on the SH3GLB1 gene are c.413C > T (p.Thr138Met) and c.646A > G (p.LysGlu) shared by the patient. According to the informatics analysis, the parents carry one mutation, and the two missense mutations in SH3GLB1 can form a compound heterozygous mutation.

PCR amplification products were analyzed by sequencing using an automatic gene analyzer 3500 of ABI, USA, and the sequencing map was analyzed and compared with a standard sequence to analyze mutation. Identification of the causative gene and causative mutation indicates that the 413 th base of the coding region of SH3GLB1 gene underwent C → T mutation and the 646 th base underwent A → G mutation (FIG. 3).

4. Experimental verification

The SH3GLB1 gene contains 9 exons, encodes a SRC homology 3 Domain protein (SH3 Domain binding GRB2 Like, endogilin B1), interacts with the pro-apoptotic member of the Bcl-2 family, Bcl-2-associated protein X (Bax) and may be involved in regulating apoptotic signaling pathways, and may also be involved in maintaining mitochondrial morphology. To date, no role for SH3GLB1 in the auditory system has been discovered. However, both the MGI database (http:// www.informatics.jax.org /) and the SHIELD database (https:// SHIELD. hms. harvard. edu/gene _ search. html) recorded that the gene was expressed in the inner ear.

To further investigate the function of SH3GLB1 in the auditory system, the inventors found that SH3GLB1 gene was widely expressed in the inner ear by immunofluorescence mapping using a mouse model.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, or materials described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

SEQUENCE LISTING

<110> general hospital of liberation military of Chinese people

Shenzhen Huada Institute of Life Sciences

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<130> PIDC3183602

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<170> PatentIn version 3.3

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Claims

1. A nucleic acid, wherein the nucleic acid is a nucleic acid,

with wild typeSH3GLB1The nucleic acid has the following mutations compared to the gene:

c.646A>G，

the nucleic acid is DNA.

2. The nucleic acid of claim 1, wherein said nucleic acid further comprises a c.413c > T mutation.

3. Use of a reagent for detecting a nucleic acid according to claim 1 or 2 for the preparation of a kit or device for the diagnosis of non-syndromic genetic deafness;

with wild typeSH3GLB1The nucleic acid has the following mutations compared to the gene: c.413C>T and/or c.646A>G。

4. The use according to claim 3, wherein the non-syndromic hereditary hearing loss is non-synthetic autosomal recessive hereditary hearing loss.

5. The use of claim 3, wherein the reagent comprises at least one of a probe specific for the nucleic acid, a primer, and a mass spectrometric detection reagent.

6. Use of a biological model carrying a nucleic acid according to claim 1 or 2 for screening a drug;

the biological model is a cell model.

7. A construct comprising the nucleic acid of claim 1 or 2.

8. A recombinant cell obtained by transforming a recipient cell with the construct of claim 7.

9. A kit for detecting non-syndromic hereditary hearing loss, comprising reagents for detecting the nucleic acid of claim 1 or 2.