HK1227057A1 - Ptprq gene mutant and application thereof - Google Patents

Abstract

The present invention discloses PTPRQ gene mutants and their applications, specifically involving isolated nucleic acids encoding PTPRQ mutants, isolated peptides, a method for screening biological samples susceptible to autosomal recessive non comprehensive hearing loss, a system for screening biological samples susceptible to autosomal recessive non comprehensive hearing loss, and a kit for screening biological samples susceptible to autosomal recessive non comprehensive hearing loss.Among them, the isolated nucleic acid encoding PTPRQ mutant has c.3125A>compared with SEQ ID NO: 1; G mutation, or c.5981A>; G mutation.By detecting the presence of this new mutant in biological samples, it is possible to effectively detect whether biological samples are susceptible to autosomal recessive non synthetic hearing loss.

Description

PTPRQ gene mutant and application thereof

Technical Field

The invention relates to a PTPRQ gene mutant and application thereof. In particular, the invention relates to isolated nucleic acids encoding PTPRQ mutants, isolated polypeptides, systems for screening biological samples susceptible to autosomal recessive and non-integrated deafness, kits, constructs, recombinant cells for screening biological samples susceptible to autosomal recessive and non-integrated deafness and methods for constructing drug screening models.

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.

Hereditary hearing loss can be classified into non-syndromic hereditary hearing loss (NSHHL) and Syndromic Hereditary Hearing Loss (SHHL) depending on whether or not symptoms accompany other tissues or organs.

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.

At present, but the causes of many patients with autosomal recessive non-synthetic deafness are unknown, so the research on the autosomal recessive non-synthetic deafness, especially the pathogenic genes thereof, is still needed.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, an object of the present invention is to provide a means for efficiently screening a biological sample susceptible to autosomal recessive non-synthetic deafness.

The present invention is completed based on the following work of the inventors:

the inventor finds that the whole exome sequencing method is mostly used in the current research of the pathogenic gene and pathogenic mutation excavation. The sequencing of the whole exome is a genome analysis method which utilizes a sequence capture technology to capture and enrich DNA of the whole genome exome region and then carry out high-throughput sequencing, has lower cost relative to the sequencing of the genome, has greater advantages on researching SNP, Indel and the like of known genes, and also plays an important role in the discovery and research of pathogenic new genes. There are many cases of finding the mendelian genetic disease causing gene by exome sequencing, for example, Sarah et al successfully locates gene DHODH of mile syndrome by exome sequencing in 2009 to find a causing mutation of miller syndrome, which is the first successful application of exome sequencing; china Wang et al discovered a new mutant gene TGM6 of cerebellar ataxia by exome sequencing. Exon sequencing technology is a current hotspot technology, and greatly promotes the progress of disease research. Aiming at the characteristics of unclear gene research on sensorineural deafness, more deafness-related genes, small family scale and the like, the exome sequencing technology is the most effective method for searching pathogenic genes.

Therefore, aiming at one autosomal recessive non-synthetic deafness (ARNSHL, also called as autosomal recessive non-synthetic hearing loss) patient family collected by self, the inventor carries out pathogenic mutation mining and verification by exome sequencing, linkage analysis and Sanger sequencing verification, finally determines 2 new pathogenic mutation sites of the autosomal recessive non-synthetic deafness, namely c.3125A > G mutation and c.5981A > G mutation of PTPRQ gene, and the compound heterozygous mutation of c.3125A > G and c.5981A > G leads to the occurrence of the autosomal recessive non-synthetic deafness.

Furthermore, according to a first aspect of the invention, the invention provides an isolated nucleic acid encoding a PTPRQ mutant. According to an embodiment of the invention, the nucleic acid is identical to SEQ ID NO: 1 compared to 1, having a c.3125a > G mutation, or a c.5981a > G mutation. According to the embodiment of the invention, the inventor determines the mutants of the PTPRQ gene, and the novel mutants are closely related to the pathogenesis of autosomal recessive non-synthetic deafness, so that whether the biological sample is susceptible to autosomal recessive non-synthetic deafness can be effectively detected by detecting whether the mutants coexist in the biological sample.

According to a second aspect of the invention, an isolated polypeptide is provided. According to an embodiment of the present invention, the sequence shown in seq id NO: 2, the isolated polypeptide has a p.d1042g mutation, or a p.e1994g mutation. By detecting whether the polypeptide is simultaneously expressed in the biological sample, whether the biological sample is susceptible to autosomal recessive nonsynthesis type deafness can be effectively detected.

According to a third aspect of the present invention, a system for screening a biological sample susceptible to autosomal recessive non-synthetic deafness is presented. According to an embodiment of the invention, the system comprises: a nucleic acid extraction device for extracting a nucleic acid sample from the biological sample; a nucleic acid sequence determining device connected with the nucleic acid extracting device and used for analyzing the nucleic acid sample so as to determine the nucleic acid sequence of the nucleic acid sample; a judging means connected to the nucleic acid sequence determining means so as to compare the nucleic acid sequence of the nucleic acid sample or a complementary sequence thereof with the nucleic acid sequence of SEQ ID NO: 1, and judging whether the biological sample is susceptible to autosomal recessive nonsynthetic deafness or not by judging whether the biological sample has compound heterozygous mutation of c.3125A > G and c.5981A > G. By utilizing the system, biological samples susceptible to autosomal recessive nonsynthetic deafness can be effectively screened.

According to a fourth aspect of the present invention, a kit for screening a biological sample susceptible to autosomal recessive non-synthetic deafness is presented. According to an embodiment of the invention, the kit comprises: suitable for detecting a peptide corresponding to SEQ ID NO: 1, and a PTPRQ gene mutant having a c.3125a > G mutation compared to SEQ ID NO: 1 compared to a PTPRQ gene mutant with a c.5981a > G mutation. By using the kit provided by the embodiment of the invention, the biological sample susceptible to autosomal recessive non-synthetic deafness can be effectively screened.

According to a fifth aspect of the invention, the invention also proposes a construct. According to an embodiment of the invention, the construct comprises the isolated nucleic acid encoding a PTPRQ mutant as described above. It is noted that by "the construct comprises the isolated nucleic acid encoding a PTPRQ mutant as described above" it is meant that the construct of the invention comprises a nucleotide sequence which is identical to the nucleotide sequence of SEQ ID NO: 1, or a nucleic acid sequence comprising a mutant PTPRQ gene having a c.1229delt mutation compared to SEQ ID NO: 1, or a nucleic acid sequence comprising both of the PTPRQ gene mutants having the c.5840c > G mutation. Therefore, the recombinant cell obtained by transforming the receptor cell by using the construct can be effectively used for screening the medicines for treating autosomal recessive nonsynthesized deafness.

According to a sixth aspect of the invention, the invention also provides a recombinant cell. According to an embodiment of the invention, the recombinant cell is obtained by transforming a recipient cell with the construct described above. According to some embodiments of the present invention, the recombinant cells of the present invention can be used to effectively screen drugs for treating autosomal recessive and non-synthetic deafness.

According to the seventh aspect of the invention, the invention also provides a method for constructing a drug screening model. According to an embodiment of the invention, the method comprises: allowing at least a portion of the cells of the animal to simultaneously express an amino acid sequence identical to SEQ ID NO: 1, and a nucleic acid having a c.3125a > G mutation as compared to SEQ ID NO: 1 to a nucleic acid having a c.5981A > G mutation. Therefore, the drug screening model of the invention can effectively screen the drugs for treating autosomal recessive non-synthetic deafness.

The two new pathogenic loci of the autosomal recessive non-synthetic deafness pathogenic gene PTPRQ discovered by the invention can be used for early screening carriers of autosomal recessive non-synthetic deafness pathogenic mutation, and then early intervention treatment is carried out before the carriers are attacked; the kit can also be used for molecular diagnosis of autosomal recessive non-synthetic deafness patients and differential diagnosis of related diseases, is quick, accurate, efficient, simple and convenient, has high early diagnosis rate, and can provide scientific basis for early diagnosis and differential diagnosis of autosomal recessive non-synthetic deafness and development of autosomal recessive non-synthetic deafness treatment drugs according to detection results.

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 shows a schematic diagram of a system and its components for screening a biological sample susceptible to autosomal recessive non-integrated deafness according to an embodiment of the present invention, wherein,

FIG. 1I is a schematic diagram of a system for screening a biological sample susceptible to autosomal recessive non-integrated deafness according to an embodiment of the present invention,

FIG. 1II is a schematic view of a nucleic acid extracting apparatus according to an embodiment of the present invention,

FIG. 1III is a schematic view of a nucleic acid sequence determination apparatus according to an embodiment of the present invention;

figure 2 shows a family diagram of ARNSHL patient families according to one embodiment of the invention;

FIG. 3 shows pure tone audiometry results for two patients in the ARNSHL patient family of FIG. 2, according to an embodiment of the present invention; and

figure 4 shows a representative Sanger sequencing validation peak plot of the PTPRQ gene c.3125a > G, c.5981a > G mutation sites for all family members in the ARNSHL patient family shown in figure 2, according to one embodiment of the present invention.

Detailed Description

The following describes embodiments of the present invention in detail. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

PTPRQ gene mutant

It should be noted that the invention uses the whole exon sequencing technology to sequence 4 samples (two diseased two normal) in an autosomal recessive non-integrated hearing loss (arnhl) family, and simultaneously uses 328 randomly selected extra-family normal persons as a control to carry out the extended analysis of the variation. Analysis revealed that of the 328 normal control samples, only 2 samples had the c.3125a > G mutation, whereas none of the c.5981a > G mutations were present in these samples. It could thus be demonstrated that mutations in the PTPRQ gene can lead to autosomal recessive deafness: PTPRQ c.3125a > G p.d1042g (maternal allele) and c.5981a > G p.e1994g (paternal allele) complex heterozygous mutations are mutations that cause autosomal recessive nonsynthetic deafness (ARNSHL).

Thus, according to a first aspect of the invention, there is provided an isolated nucleic acid encoding a PTPRQ mutant. According to embodiments of the invention, the sequence of SEQ ID NO: 1, or a c.5981a > G mutation. The expression "nucleic acid encoding a PTPRQ mutant" as used herein refers to a nucleic acid substance corresponding to a gene encoding a PTPRQ mutant, i.e., the type of nucleic acid is not particularly limited, and may be any polymer comprising deoxyribonucleotides and/or ribonucleotides corresponding to a gene encoding a PTPRQ mutant, including but not limited to DNA, RNA, or cDNA. According to a particular embodiment of the invention, the nucleic acid encoding a PTPRQ mutant described above is DNA. According to the embodiment of the invention, the inventor determines new mutants of PTPRQ genes, and the mutants are closely related to the pathogenesis of autosomal recessive non-integrated deafness, so that whether a biological sample is susceptible to autosomal recessive non-integrated deafness or not can be effectively detected by detecting whether the mutants coexist in the biological sample, and whether the organism is susceptible to autosomal recessive non-integrated deafness or not can be effectively predicted by detecting whether the mutants coexist in the organism or not.

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 is made to SEQ ID NO: 1, actually including its complement. One skilled in the art will also appreciate that one strand may be used to detect the other strand and vice versa.

The nucleic acid encoding the PTPRQ mutant is a new pathogenic mutation of a pathogenic gene PTPRQ of autosomal recessive nonsynthetic deafness, which is determined by exome sequencing and linkage analysis combined with Sanger sequencing verification by the inventor of the application. These pathogenic mutation sites have not been mentioned in the prior art.

The cDNA of the wild-type PTPRQ gene had the nucleotide sequence shown below (the transcript of RefSeq, No. NM — 001145026.1 was used):

ATGGATTTTCTTATCATTTTTCTTTTACTTTTTATTGGGACTTCAGAGACACAGGTTGATGTTTCCAATGTCGTTCCTGGTACTAGGTACGATATAACCATCTCTTCAATTTCTACAACATACACCTCACCTGTTACTAGAATAGTGACAACAAATGTAACAAAACCAGGGCCTCCAGTCTTCCTAGCCGGGGAAAGAGTCGGATCTGCTGGGATTCTTCTGTCTTGGAATACACCACCTAATCCAAATGGAAGGATTATATCTTACATTGTCAAATATAAGGAAGTTTGTCCGTGGATGCAAACAGTATATACACAAGTCAGATCAAAGCCAGACAGTCTGGAAGTTCTTCTTACTAATCTTAATCCTGGAACAACATATGAAATTAAGGTTGCTGCTGAAAACAGTGCTGGCATTGGAGTGTTTAGTGATCCATTTCTCTTCCAAACTGCAGAAAGTGCTCCAGGAAAAGTGGTGAATCTCACAGTTGAGGCCTACAACGCTTCAGCAGTTAAGCTGATTTGGTATTTACCTCGGCAACCAAATGGCAAAATTACCAGCTTCAAGATTAGTGTCAAGCATGCCAGAAGTGGGATAGTAGTGAAAGATGTCTCAATCAGAGTAGAGGACATTTTGACTGGGAAATTGCCAGAATGCAATGAGAATAGTGAATCTTTTTTATGGAGTACAGCCAGCCCTTCTCCAACCCTTGGTAGAGTTACACCTCCATCGCGTACCACACATTCATCAAGCACGTTGACACAGAATGAGATCAGCTCTGTGTGGAAAGAGCCTATCAGTTTTGTAGTGACACACTTGAGACCTTATACAACATATCTTTTTGAAGTTTCAGCTGCTACAACTGAAGCAGGTTATATTGATAGTACGATTGTCAGAACACCAGAATCAGTGCCTGAAGGACCACCACAAAACTGCGTAACAGGCAACATCACAGGAAAGTCCTTTTCAATTTTATGGGACCCACCAACTATAGTAACAGGGAAATTTAGTTATAGAGTTGAATTATATGGACCATCAGGTCGCATTTTGGATAACAGCACAAAAGACCTCAAGTTTGCATTCACTAACCTAACACCATTTACAATGTATGATGTCTATATTGCGGCTGAAACCAGTGCAGGGACTGGGCCCAAGTCAAATATTTCAGTATTCACTCCACCAGATGTTCCAGGGGCAGTGTTTGATTTACAACTTGCAGAGGTAGAATCCACGCAAGTAAGAATTACTTGGAAGAAACCACGACAACCAAATGGAATTATTAACCAATACCGAGTGAAAGTGCTAGTTCCAGAGACAGGAATAATTTTGGAAAATACTTTGCTCACTGGAAATAATGAGTATATAAATGACCCCATGGCTCCAGAAATTGTGAACATAGTAGAGCCAATGGTAGGATTATATGAGGGTTCAGCAGAGATGTCGTCTGACCTTCACTCACTTGCTACATTTATATATAACAGCCATCCAGATAAAAACTTTCCTGCAAGGAATAGAGCTGAAGACCAGACTTCACCAGTTGTAACTACAAGGAATCAGTATATTACTGACATTGCAGCTGAACAGCTGTCTTATGTTATCAGGAGACTTGTACCTTTCACTGAGCACATGATTAGTGTATCTGCTTTCACCATCATGGGAGAAGGACCACCAACAGTTCTCAGTGTTAGGACACGTCAGCAAGTGCCAAGCTCCATTAAAATTATAAACTATAAAAATATTAGTTCTTCATCTATTTTGTTATATTGGGATCCTCCAGAATATCCCAATGGAAAAATAACTCACTATACGATTTATGCAATGGAATTGGATACAAACAGAGCATTCCAGATAACTACCATAGATAACAGCTTTCTCATAACAGGGTTAAAGAAATACACAAAATACAAAATGAGAGTGGCAGCCTCAACCCACGTTGGAGAAAGTTCTTTGTCTGAAGAAAATGACATCTTTGTGAGAACTTCAGAAGATGAACCGGAATCATCACCTCAAGATGTCGAAGTAATTGATGTTACCGCAGATGAAATAAGGTTGAAGTGGTCACCACCCGAAAAGCCCAATGGGATCATTATTGCTTATGAAGTGCTATATAAAAATATAGATACTTTATATATGAAGAACACATCAACAACAGACATAATATTAAGGAACTTAAGACCTCACACCCTCTATAACATTTCTGTAAGGTCTTACACCAGATTTGGTCATGGCAATCAGGTATCTTCTTTACTCTCTGTAAGGACTTCGGAGACTGTGCCTGATAGTGCACCAGAAAATATCACTTACAAAAATATTTCTTCTGGAGAGATTGAGCTATCATTCCTTCCCCCAAGTAGTCCCAATGGAATCATACAAAAATATACAATTTATCTCAAGAGAAGTAATGGAAATGAGGAAAGAACTATAAATACAACCTCTTTAACCCAAAACATTAAAGTACTGAAGAAATATACCCAATATATCATTGAGGTGTCTGCTAGTACACTCAAAGGTGAAGGAGTTCGGAGTGCTCCCATAAGTATACTGACGGAGGAAGATGCTCCTGATTCTCCCCCTCAAGACTTCTCTGTAAAACAGTTGTCTGGTGTCACGGTGAAGTTGTCATGGCAACCACCCCTGGAGCCAAATGGAATTATCCTTTATTACACAGTTTATGTCTGGAATAGATCATCATTAAAAACTATTAATGTCACTGAAACATCATTGGAGTTATCAGATTTGGATTATAATGTTGAATACAGTGCTTATGTAACAGCTAGCACCAGATTTGGTGATGGGAAAACAAGAAGCAATATCATTAGCTTTCAAACACCAGAGGGAGCACCAAGCGATCCTCCCAAAGATGTTTATTATGCAAACCTCAGTTCTTCATCAATAATTCTTTTCTGGACACCTCCTTCAAAACCTAATGGGATTATACAATATTACTCTGTTTATTACAGAAATACTTCAGGTACTTTTATGCAGAATTTTACACTCCATGAAGTAACCAATGACTTTGACAATATGACTGTATCCACAATTATAGATAAACTGACAATATTCAGCTACTATACATTTTGGTTAACAGCAAGTACTTCAGTTGGAAATGGGAATAAAAGCAGTGACATCATTGAAGTATACACAGATCAAGACATACCTGAAGGGTTTGTTGGAAACCTGACTTACGAATCCATTTCGTCAACTGCAATAAATGTAAGCTGGGTCCCACCGGCTCAACCAAACGGTCTAGTCTTCTACTATGTTTCACTGATCTTACAGCAGACTCCTCGCCATGTGAGACCACCTCTTGTTACATATGAGAGAAGCATATATTTTGATAATCTGGAAAAATACACTGATTATATATTAAAAATTACTCCATCAACAGAAAAGGGATTCTCTGATACCTATACTGCCCAGCTATACATCAAGACTGAAGAAGATGTCCCAGAAACTTCACCAATAATCAACACTTTTAAAAACCTTTCCTCTACCTCAGTTCTCTTATCATGGGATCCCCCAGTAAAGCCAAATGGTGCAATAATAAGTTATGATTTAACTTTACAAGGACCAAATGAAAATTATTCTTTCATTACTTCTGATAATTACATAATATTGGAAGAGCTTTCACCATTTACATTATATAGCTTTTTTGCTGCCGCAAGAACTAGAAAAGGACTTGGTCCTTCCAGTATTCTTTTCTTTTACACAGATGAGTCAGTGCCGTTAGCACCTCCACAAAATTTGACTTTAATCAACTGTACTTCAGACTTTGTATGGCTGAAATGGAGCCCAAGTCCTCTTCCAGGTGGTATTGTTAAAGTATATAGTTTTAAAATTCATGAACATGAAACTGACACTATATATTATAAGAATATATCAGGATTTAAAACTGAAGCCAAACTTGTTGGACTGGAACCAGTCAGCACCTACTCTATCCGTGTATCTGCGTTCACCAAAGTTGGAAATGGCAATCAATTTAGTAATGTAGTAAAATTCACAACCCAAGAATCAGTTCCAGATGTCGTGCAGAATATGCAGTGCATGGCAACTAGCTGGCAGTCAGTTTTAGTGAAATGGGATCCACCCAAAAAGGCAAATGGAATAATAACGCAGTATATGGTAACAGTTGAAAGGAATTCTACAAAAGTTTCTCCCCAAGATCACATGTACACTTTCATAAAGCTTCTTGCCAATACCTCATATGTCTTTAAAGTAAGAGCTTCAACCTCAGCTGGTGAAGGTGATGAAAGCACATGCCATGTCAGCACACTACCTGAAACAGTTCCCAGTGTTCCCACAAATATTGCTTTTTCTGATGTTCAGTCAACTAGTGCAACATTGACATGGATAAGACCTGACACTATCCTTGGCTACTTTCAAAATTACAAAATTACCACTCAACTTCGTGCTCAAAAATGCAAAGAATGGGAATCCGAAGAATGTGTTGAATATCAAAAAATTCAATACCTCTATGAAGCTCACTTAACTGAAGAGACAGTATATGGATTAAAGAAATTTAGATGGTATAGATTCCAAGTGGCTGCCAGCACCAATGCTGGCTATGGCAATGCTTCAAACTGGATTTCTACAAAAACTCTGCCTGGCCCTCCAGATGGTCCTCCTGAAAATGTTCATGTAGTAGCAACATCACCTTTTAGCATCAGCATAAGCTGGAGTGAACCTGCTGTCATTACTGGACCAACATGTTATCTGATTGATGTCAAATCGGTAGATAATGATGAATTTAATATATCCTTCATCAAGTCAAATGAAGAAAATAAAACCATAGAAATTAAAGATTTAGAAATATTCACAAGGTATTCTGTAGTGATCACTGCATTTACTGGGAACATTAGTGCTGCATATGTAGAAGGGAAGTCAAGTGCTGAAATGATTGTTACTACTTTAGAATCAGCCCCAAAGGACCCACCTAACAACATGACATTTCAGAAGATACCAGATGAAGTTACAAAATTTCAATTAACGTTCCTTCCTCCTTCTCAACCTAATGGAAATATCCAAGTATATCAAGCTCTGGTTTACCGAGAAGATGATCCTACTGCTGTCCAGATTCACAACCTCAGTATTATACAGAAAACCAACACATTCGTCATTGCAATGCTAGAAGGACTAAAAGGTGGACATACATACAATATCAGTGTTTACGCAGTCAATAGTGCTGGTGCAGGTCCAAAGGTTCCGATGAGAATAACCATGGATATCAAAGCTCCAGCACGACCAAAAACCAAACCAACCCCTATTTATGATGCCACAGGAAAACTGCTTGTGACTTCAACAACAATTACAATCAGAATGCCAATATGTTACTACAGTGATGATCATGGACCAATAAAAAATGTACAAGTGCTTGTGACAGAAACAGGAGCTCAGCATGATGGAAATGTAACAAAGTGGTATGATGCATATTTTAATAAAGCAAGGCCATATTTTACAAATGAAGGCTTTCCTAACCCTCCATGTACAGAAGGAAAGACAAAGTTTAGTGGCAATGAAGAAATCTACATCATAGGTGCTGATAATGCATGCATGATTCCTGGCAATGAAGACAAAATTTGCAATGGACCACTGAAACCAAAAAAGCAATACTTATTTAAATTTAGAGCTACAAATATTATGGGACAATTTACTGACTCTGATTATTCTGACCCTGTTAAGACTTTAGGGGAAGGACTTTCAGAAAGAACCGTAGAGATCATTCTTTCCGTCACTTTGTGTATCCTTTCAATAATTCTCCTTGGAACAGCTATTTTTGCATTTGCAAGAATTCGACAGAAGCAGAAAGAAGGTGGCACATACTCTCCTCAGGATGCAGAAATTATTGACACTAAATTGAAGCTGGATCAGCTCATCACAGTGGCAGACCTGGAACTGAAGGACGAGAGATTAACGCGGTTACTTAGTTATAGAAAATCCATCAAGCCAATAAGCAAGAAATCCTTCCTGCAACATGTTGAAGAGCTTTGCACAAACAACAACCTAAAGTTTCAAGAAGAATTTTCGGAATTACCAAAATTTCTTCAGGATCTTTCTTCAACTGATGCTGATCTGCCTTGGAATAGAGCAAAAAACCGCTTCCCAAACATAAAACCATATAATAATAACAGAGTAAAGCTGATAGCTGACGCTAGTGTTCCAGGTTCGGATTATATTAATGCCAGCTATATTTCTGGTTATTTATGTCCAAATGAATTTATTGCTACTCAAGGTCCACTACCAGGAACAGTTGGAGATTTTTGGAGAATGGTGTGGGAAACCAGAGCAAAAACATTAGTAATGCTAACACAGTGTTTTGAAAAAGGACGGATCAGATGCCATCAGTATTGGCCAGAGGACAACAAGCCAGTTACTGTCTTTGGAGATATAGTGATTACAAAGCTAATGGAGGATGTTCAAATAGATTGGACTATCAGGGATCTGAAAATTGAAAGGCATGGGGATTGCATGACTGTTCGACAGTGTAACTTTACTGCCTGGCCAGAGCATGGGGTTCCTGAGAACAGCGCCCCTCTAATTCACTTTGTGAAGTTGGTTCGAGCAAGCAGGGCACATGACACCACACCTATGATTGTTCACTGCAGTGCTGGAGTTGGAAGAACTGGAGTTTTTATTGCTCTGGACCATTTAACACAACATATAAATGACCATGATTTTGTGGATATATATGGACTAGTAGCTGAACTGAGAAGTGAAAGAATGTGCATGGTGCAGAATCTGGCACAGTATATCTTTTTACACCAGTGCATTCTGGATCTCTTATCAAATAAGGGAAGTAATCAGCCCATCTGTTTTGTTAACTATTCAGCACTTCAGAAGATGGACTCTTTGGACGCCATGGAAGGTGATGTTGAGCTTGAATGGGAAGAAACCACTATGTAAATATTCAGACCAAAGGATACAATTGGAAGAGATTTTTAAATCCCAGGGGCCAAAGTTACCCCCTCATTCTTCCGAATTGAAATGTGCAACCTTAAAGAAATATCTATGCTTCTCTCACTGTGCCTTTCCAAACGGATTGAACATTTTAAGACTAGTTCTTGAAAATAGCTAATACAGAATAATTATTTGTTTTGTACAGAATAAATATTATGCATTTTAAATGCTTAAGAAAAGACATCCCATATGTTTTTGAAGTCCTCCATATTTTGGAATAAGCCAAATAGAAAATTATTATTATATTAGCATTAATGTTTCAATGTGAATTTTCCCTATGTATTGGATTTAATTTTGAGCAAAAGTTGTAAATGTTGATTCAGTAGTGTTGTTTTGGCTTACAGGGTATTGATGTTTCTTGTGGATAATTTCCAGGACTGTCATAATGATCTGTACTTCCATGTACACCCCTGTGTTTTGAATCCTCTGTTTTATGAGTGCTGAGATATCATCTCATGATCCCGAACAGCTGAACAGTAACCCCCTGACACTGCAGGGATTACTTGGCCTTTATACAACACACAGTAGCTCTTCAGGGACACTTAGGGCTATTTAATTTGCATTGTGATCTTCAGTTTGAGAACCTTAAAAGAAAAATTAAAAGTGCAATTGCACACATGAAATTACAGAGTACCATTCTAGCAAACCTACATTTGTAAACTTTAAAACACAAGTTTTACCCCCTGTATTGTATATTCAAATATATAGTAAATGTATCAGAGTATTTGCCCATTAGATATAGTCAACCTAATATTAACAATTCTGAAGAGTTTCTTCAGCAAAAATGTATCAAAAGAGTAATAAAAACACTGTGCGTGTTTCAAGCTTGTAAACCAATGATGTGCTGCTGTGGTGCCAACAGAGACTTCCAAATGGATTATGTTAAATGGCCGTCATTTCATTTCCCAAGGTTGATTTTGAGCAGTATACTTGGTGGAACTGAAAACAAAGAAATTAACCATGTATAGCAAATTCAAGGTTTCTTTATAGAAAATCTTTCAGCCTCCATCTTATTAAATAGTGACAATGTGGTAAGTTTTGAATTACATGAACTCATTTTGTCATAGATTTCAATTAAGAGTAATAAATAGTATTAATTATTCTCTTCTATG (SEQ ID NO: 1), which encodes a protein having the amino acid sequence shown below:

MDFLIIFLLLFIGTSETQVDVSNVVPGTRYDITISSISTTYTSPVTRIVTTNVTKPGPPVFLAGERVGSAGILLSWNTPPNPNGRIISYIVKYKEVCPWMQTVYTQVRSKPDSLEVLLTNLNPGTTYEIKVAAENSAGIGVFSDPFLFQTAESAPGKVVNLTVEAYNASAVKLIWYLPRQPNGKITSFKISVKHARSGIVVKDVSIRVEDILTGKLPECNENSESFLWSTASPSPTLGRVTPPSRTTHSSSTLTQNEISSVWKEPISFVVTHLRPYTTYLFEVSAATTEAGYIDSTIVRTPESVPEGPPQNCVTGNITGKSFSILWDPPTIVTGKFSYRVELYGPSGRILDNSTKDLKFAFTNLTPFTMYDVYIAAETSAGTGPKSNISVFTPPDVPGAVFDLQLAEVESTQVRITWKKPRQPNGIINQYRVKVLVPETGIILENTLLTGNNEYINDPMAPEIVNIVEPMVGLYEGSAEMSSDLHSLATFIYNSHPDKNFPARNRAEDQTSPVVTTRNQYITDIAAEQLSYVIRRLVPFTEHMISVSAFTIMGEGPPTVLSVRTRQQVPSSIKIINYKNISSSSILLYWDPPEYPNGKITHYTIYAMELDTNRAFQITTIDNSFLITGLKKYTKYKMRVAASTHVGESSLSEENDIFVRTSEDEPESSPQDVEVIDVTADEIRLKWSPPEKPNGIIIAYEVLYKNIDTLYMKNTSTTDIILRNLRPHTLYNISVRSYTRFGHGNQVSSLLSVRTSETVPDSAPENITYKNISSGEIELSFLPPSSPNGIIQKYTIYLKRSNGNEERTINTTSLTQNIKVLKKYTQYIIEVSASTLKGEGVRSAPISILTEEDAPDSPPQDFSVKQLSGVTVKLSWQPPLEPNGIILYYTVYVWNRSSLKTINVTETSLELSDLDYNVEYSAYVTASTRFGDGKTRSNIISFQTPEGAPSDPPKDVYYANLSSSSIILFWTPPSKPNGIIQYYSVYYRNTSGTFMQNFTLHEVTNDFDNMTVSTIIDKLTIFSYYTFWLTASTSVGNGNKSSDIIEVYTDQDIPEGFVGNLTYESISSTAINVSWVPPAQPNGLVFYYVSLILQQTPRHVRPPLVTYERSIYFDNLEKYTDYILKITPSTEKGFSDTYTAQLYIKTEEDVPETSPIINTFKNLSSTSVLLSWDPPVKPNGAIISYDLTLQGPNENYSFITSDNYIILEELSPFTLYSFFAAARTRKGLGPSSILFFYTDESVPLAPPQNLTLINCTSDFVWLKWSPSPLPGGIVKVYSFKIHEHETDTIYYKNISGFKTEAKLVGLEPVSTYSIRVSAFTKVGNGNQFSNVVKFTTQESVPDVVQNMQCMATSWQSVLVKWDPPKKANGIITQYMVTVERNSTKVSPQDHMYTFIKLLANTSYVFKVRASTSAGEGDESTCHVSTLPETVPSVPTNIAFSDVQSTSATLTWIRPDTILGYFQNYKITTQLRAQKCKEWESEECVEYQKIQYLYEAHLTEETVYGLKKFRWYRFQVAASTNAGYGNASNWISTKTLPGPPDGPPENVHVVATSPFSISISWSEPAVITGPTCYLIDVKSVDNDEFNISFIKSNEENKTIEIKDLEIFTRYSVVITAFTGNISAAYVEGKSSAEMIVTTLESAPKDPPNNMTFQKIPDEVTKFQLTFLPPSQPNGNIQVYQALVYREDDPTAVQIHNLSIIQKTNTFVIAMLEGLKGGHTYNISVYAVNSAGAGPKVPMRITMDIKAPARPKTKPTPIYDATGKLLVTSTTITIRMPICYYSDDHGPIKNVQVLVTETGAQHDGNVTKWYDAYFNKARPYFTNEGFPNPPCTEGKTKFSGNEEIYIIGADNACMIPGNEDKICNGPLKPKKQYLFKFRATNIMGQFTDSDYSDPVKTLGEGLSERTVEIILSVTLCILSIILLGTAIFAFARIRQKQKEGGTYSPQDAEIIDTKLKLDQLITVADLELKDERLTRLLSYRKSIKPISKKSFLQHVEELCTNNNLKFQEEFSELPKFLQDLSSTDADLPWNRAKNRFPNIKPYNNNRVKLIADASVPGSDYINASYISGYLCPNEFIATQGPLPGTVGDFWRMVWETRAKTLVMLTQCFEKGRIRCHQYWPEDNKPVTVFGDIVITKLMEDVQIDWTIRDLKIERHGDCMTVRQCNFTAWPEHGVPENSAPLIHFVKLVRASRAHDTTPMIVHCSAGVGRTGVFIALDHLTQHINDHDFVDIYGLVAELRSERMCMVQNLAQYIFLHQCILDLLSNKGSNQPICFVNYSALQKMDSLDAMEGDVELEWEETTM(SEQ ID NO：2)。

two PTPRQ gene mutants discovered by the inventors, one of which is homologous to SEQ ID NO: 1, has a c.3125A > G mutation, i.e., the 3125 th base A in the cDNA of the PTPRQ gene mutant is mutated to G relative to the wild-type PTPRQ gene, and thus the encoded product has a p.D1042G mutation compared to the wild-type PTPRQ (SEQ ID NO: 2), i.e., the mutation is a missense mutation due to the c.3125A > G mutation, resulting in the 1042 th amino acid aspartic acid being mutated to glycine.

And another PTPRQ gene mutant, which has a sequence identical to SEQ ID NO: 1, has c.5981A > G mutation, i.e. the 5981 th base A in the cDNA of the PTPRQ gene mutant is mutated to G relative to the wild-type PTPRQ gene, therefore, the coded product has missense mutation of p.E1994G, i.e. the 1994 th amino acid is mutated from glutamic acid to glycine compared with the wild-type PTPRQ (SEQ ID NO: 2).

The invention discloses two new mutations of the known deafness gene PTPRQ, and proves that the patient has the symptom of autosomal recessive nonsynthesized deafness caused by the compound heterozygous mutation of c.3125A > G and c.5981A > G of the PTPRQ gene, namely the patient is the molecular cause of autosomal recessive hereditary deafness.

According to a second aspect of the invention, an isolated polypeptide is provided. According to an embodiment of the invention, the isolated polypeptide has a p.d1042g mutation, or a p.e1994g mutation, compared to the wild-type PTPRQ. According to some embodiments of the invention, the polypeptide having the p.d1042g mutation is encoded by the aforementioned isolated nucleic acid encoding a PTPRQ gene mutant having the c.3125a > G mutation, and the polypeptide having the p.e1994g mutation is encoded by the aforementioned isolated nucleic acid encoding a PTPRQ gene mutant having the c.5981a > G mutation. Whether the biological sample is susceptible to autosomal recessive non-integrated deafness can be effectively detected by detecting whether the polypeptides are expressed in the biological sample at the same time, and whether the biological sample is susceptible to autosomal recessive non-integrated deafness can be effectively predicted by detecting whether the polypeptides exist in the biological sample at the same time.

System and kit for screening biological samples susceptible to autosomal recessive non-integrated deafness

According to a third aspect of the present invention, the present invention provides a system capable of efficiently carrying out the above-mentioned method for screening a biological sample susceptible to autosomal recessive non-integrated deafness.

Referring to fig. 1, the system 1000 for screening a biological sample susceptible to autosomal recessive non-integrated deafness according to an embodiment of the present invention includes a nucleic acid extracting apparatus 100, a nucleic acid sequence determining apparatus 200, and a determining apparatus 300.

According to an embodiment of the present invention, the nucleic acid extraction apparatus 100 is used to extract a nucleic acid sample 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 whether the PTPRQ of the biological sample has a mutation 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, subcutaneous tissue, preferably peripheral blood. Therefore, the sampling and the detection can be conveniently carried out, and the efficiency of screening the biological sample susceptible to autosomal recessive non-synthetic deafness can be further improved. The term "nucleic acid sample" as used herein is to be understood broadly according to embodiments of the present invention and may be any sample that reflects the presence or absence of a mutation in PTPRQ in a biological sample, such as whole genomic DNA extracted directly from the biological sample, a portion of the whole genome that includes the PTPRQ coding sequence, 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 various information of the biological sample can be determined simultaneously, so that the efficiency of screening the biological sample susceptible to autosomal recessive and non-synthetic deafness can be improved. In addition, according to an embodiment of the present invention, the type of the nucleic acid sample is not particularly limited, and for using RNA as the nucleic acid sample, the nucleic acid extraction apparatus further includes an RNA extraction unit 101 and a reverse transcription unit 102, wherein the extraction unit 101 is used for extracting the RNA sample from the biological sample, and the reverse transcription unit 102 is connected to the RNA extraction unit 101 for performing a reverse transcription reaction on the RNA sample to obtain a cDNA sample, and the obtained cDNA sample constitutes the nucleic acid sample. Therefore, the efficiency of screening the biological sample susceptible to autosomal recessive non-synthetic deafness by using RNA as a nucleic acid sample can be further improved.

According to an embodiment of the present invention, the nucleic acid sequence determining apparatus 200 is connected to the nucleic acid extracting apparatus 100, and is configured to analyze the nucleic acid sample to determine the nucleic acid sequence of the 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, sequencing methods may be used to determine the nucleic acid sequence of a nucleic acid sample. Thus, according to one embodiment of the present invention, the nucleic acid sequence determination apparatus 200 may further include: a library construction unit 201 and a sequencing unit 202. The library construction unit 201 is used for constructing a nucleic acid sequencing library aiming at a nucleic acid sample; the sequencing unit 202 is connected to the library construction unit 201 and is configured to sequence the nucleic acid sequencing library to obtain a sequencing result consisting of a plurality of sequencing data.

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 platforms, 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 using sequencing data (reads) obtained by sequencing a nucleic acid sample, as long as the nucleic acid sequences contain coding sequences corresponding to PTPRQ.

In addition, according to embodiments of the present invention, a nucleic acid sample can be screened to enrich for PTPRQ exons, and the screening enrichment can be performed before, during, or after the construction of a sequencing library. Thus, the library constructing unit 201 may further comprise a PCR amplification module (not shown in the figure) in which PTPRQ exon-specific primers are disposed so as to perform PCR amplification on the nucleic acid sample using the PTPRQ exon-specific primers. Therefore, PTPRQ exons can be enriched through PCR amplification, so that the efficiency of screening biological samples susceptible to autosomal recessive nonsynthesis type deafness can be further improved. According to a specific embodiment of the invention, the PTPRQ gene exon-specific primer has the sequence as shown in SEQ ID NO: 3-4; the PTPRQ gene exon specific primer has the nucleotide sequence shown as SEQ ID NO: 5-6:

mutations	Upstream primer (5 '→ 3', SEQ ID NO:)	Downstream primer (5 '→ 3', SEQ ID NO:)
			c.3125A>G	TGTCGATTTTCCTAAAACAACAT(3)	GCAGTTTTCTTGAACAGAAGAGG(4)
c.5981A>G	TGATTTTGGGGATGTCCATT(5)	TCAAGGCCAGAGTCCTTCAT(6)

The inventors surprisingly found that amplification of PTPRQ exons can be accomplished significantly efficiently in a PCR reaction system by using the above primers. Note that, these SEQ ID NOs: 3-6 are unexpectedly obtained by the inventors of the present invention after a hard work.

According to embodiments of the present invention, the apparatus that may be used to perform sequencing is not particularly limited. According to embodiments of the present invention, second generation sequencing platforms may be employed, as well as third generation and fourth generation or more advanced sequencing platforms. According to a specific example of the present invention, the sequencing unit 202 may be at least one selected from the group consisting of hipseq 2000, 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 are improved.

According to an embodiment of the present invention, the judging means 300 is connected to the nucleic acid sequence determining means 200, and is adapted to compare the nucleic acid sequence of the nucleic acid sample with the nucleic acid sequence of SEQ ID NO: 1 whether the biological sample is susceptible to autosomal recessive nonsynthetic deafness. Therefore, the system can be used for effectively screening the biological samples susceptible to autosomal recessive non-synthetic deafness.

Specifically, the nucleic acid sample is a nucleic acid sample based on the nucleotide sequence of SEQ ID NO: 1, judging whether the biological sample is susceptible to autosomal recessive nonsynthetic deafness or not by judging whether the biological sample has compound heterozygous mutation of c.3125A > G and c.5981A > G. As previously mentioned, according to one embodiment of the invention, the nucleic acid sequence of the nucleic acid sample is identical to SEQ ID NO: 1, a compound heterozygous mutation with c.3125A > G and c.5981A > G is an indicator that the biological sample is susceptible to autosomal recessive nonsynthetic deafness. According to an embodiment of the invention, the nucleic acid sequence is identical to SEQ ID NO: 1, the device for performing the comparison is not particularly limited, and may be operated by any conventional software, and according to the specific example of the present invention, the comparison may be performed by SOAP software.

According to a fourth aspect of the present invention, a kit for screening a biological sample susceptible to autosomal recessive non-synthetic deafness is presented. According to an embodiment of the present invention, the kit for screening a biological sample susceptible to autosomal recessive non-integrated deafness comprises: suitable for detecting a peptide corresponding to SEQ ID NO: 1, and a PTPRQ gene mutant having a c.3125a > G mutation compared to SEQ ID NO: 1 compared to a PTPRQ gene mutant with a c.5981a > G mutation. By utilizing the kit provided by the embodiment of the invention, the biological sample susceptible to autosomal recessive nonsynthetic deafness can be effectively screened by screening the biological sample in which the two PTPRQ gene mutants exist simultaneously. As used herein, the term "suitable for detecting a mutation in SEQ ID NO: 1, and a PTPRQ gene mutant having a c.3125a > G mutation compared to SEQ ID NO: 1 in comparison with a reagent for detecting a mutant PTPRQ gene having a mutation of c.5981A > G "is to be understood in a broad sense, i.e., a reagent for detecting genes encoding two PTPRQ mutants, or a reagent for detecting a polypeptide of a PTPRQ mutant, and for example, an antibody recognizing a specific site may be used. According to one embodiment of the invention, the agent is a nucleic acid probe or primer, preferably directed against a nucleic acid sequence identical to SEQ ID NO: 1, and the nucleic acid probe or primer has the nucleotide sequence shown as SEQ ID NO: 3-4; for sequences similar to SEQ ID NO: 1, and the nucleic acid probe or primer has the nucleotide sequence shown in SEQ ID NO: 5-6. Therefore, the biological sample susceptible to autosomal recessive nonsynthetic deafness can be efficiently screened.

It is to be noted that the features and advantages described in the systematic section of the screening of biological samples susceptible to autosomal recessive and non-synthetic deafness hereinbefore are equally applicable to kits for screening biological samples susceptible to autosomal recessive and non-synthetic deafness and will not be described in detail herein.

Construct and recombinant cell

According to a fifth aspect of the invention, the invention also proposes a construct. According to an embodiment of the invention, the construct comprises the isolated nucleic acid encoding a PTPRQ mutant as described above. It is noted that by "the construct comprises the isolated nucleic acid encoding a PTPRQ mutant as described above" it is meant that the construct of the invention comprises a nucleotide sequence which is identical to the nucleotide sequence of SEQ ID NO: 1, or a nucleic acid sequence comprising a PTPRQ gene mutant with a c.3125a > G mutation compared to SEQ ID NO: 1, or both, of the nucleic acid sequences of the PTPRQ gene mutants having the c.5981a > G mutation. Therefore, the recombinant cell obtained by transforming the receptor cell by using the construct can be effectively used for screening the medicines for treating autosomal recessive nonsynthesized deafness. 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 a sixth aspect of the invention, the invention also provides a recombinant cell. According to an embodiment of the invention, the recombinant cell is obtained by transforming a recipient cell with the construct described above. Thus, the recombinant cells of the invention are capable of expressing the PTPRQ gene mutant carried by the construct. According to some embodiments of the present invention, the recombinant cells of the present invention can be used to effectively screen drugs for treating autosomal recessive and non-synthetic deafness. 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.

Method for constructing drug screening model

According to a seventh aspect of the present invention, the present invention also provides a method of constructing a drug screening model. According to an embodiment of the invention, the method comprises: allowing at least a portion of the cells of the animal to simultaneously express an amino acid sequence identical to SEQ ID NO: 1, and a nucleic acid having a c.3125a > G mutation as compared to SEQ ID NO: 1 to a nucleic acid having a c.5981A > G mutation. According to an embodiment of the invention, the animal is a mouse, pig, dog, primate. According to some embodiments of the invention, the drug screening model provided by the invention can be used for effectively screening drugs for treating autosomal recessive and non-synthetic deafness.

It should be noted that the method for constructing a drug screening model of the present invention is not particularly limited as long as at least a part of cells of an animal simultaneously express the aforementioned two PTPRQ gene mutants (a nucleic acid having a c.3125a > G mutation as compared with SEQ ID No. 1 and a nucleic acid having a c.5981a > G mutation as compared with SEQ ID No. 1). For example, the construct of the present invention described above can be transferred into a recipient animal (non-human) by gene transformation, so that at least a portion of the cells of the animal simultaneously express the two PTPRQ gene mutants described above; the PTPRQ gene of the receptor animal can generate composite heterozygous mutation of c.3125A > G and c.5981A > G by adopting methods such as CRISPR/Cas9 gene editing technology, cassette mutation, SOE PCR and the like, and the two polypeptides are effectively expressed, so that the receptor animal can generate autosomal recessive nonsynthesized deafness, and can be further effectively used for screening the medicine for treating the autosomal recessive nonsynthesized deafness, namely can be effectively used as a medicine screening model.

The invention adopts a new generation of whole-exome sequencing technology to carry out whole-genome exome sequencing analysis aiming at a family of autosomal recessive non-integrated deafness patients, thereby finding out 2 new mutation sites of autosomal recessive non-integrated deafness pathogenic genes PTPRQ. Compared with the traditional linkage analysis and candidate gene association analysis technologies, the exome sequencing technology aims at exome regions of coding proteins in a genome, has concentrated targets and higher sequencing depth and precision, can more accurately position the pathogenic genes and the mutation sites thereof of autosomal recessive non-synthetic deafness, and further clarifies the genetic sequence of the autosomal recessive non-synthetic deafnessThe molecular pathogenesis of the S syndrome provides scientific basis for developing effective early pathogenic gene screening and intervention treatment measures.

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.

Example 1 determination of autosomal recessive non-synthetic deafness-causing mutations

1. Sample collection

The family map of the inventor collected a Chinese Han nationality 2 generation autosomal recessive nonsynthetic deafness patient is shown in figure 2. As shown in fig. 2, the family contains 4 members, including 2 patients (i.e., II1, II2 in the family map), 2 normal persons in the family (i.e., parents I1, I2 of the 2 patients, both of which have no disease), and conforms to the autosomal recessive inheritance pattern. Where, o indicates a normal female, □ indicates a normal male, ■ indicates a male patient, and the arrow indicates a proband.

Wherein, the pure tone audiometry results of two patients in the family are shown in figure 3. In fig. 3, the abscissa represents the frequency of a pure tone, the ordinate represents the hearing level, and if the hearing normality threshold curve should be floated around 0, the curve goes down to represent hearing loss. As shown in FIG. 3, the left panel shows the test result of patient II1, the right panel shows the test result of patient II2, and the results show that the hearing of patients II1 and II2 are both bilateral moderate-severe sensorineural deafness.

The inventors collected peripheral blood samples from all members of the family, added EDTA for anticoagulation, and stored at-80 ℃. All blood samples were signed with informed consent.

2. DNA extraction

Taking peripheral Blood of all members of the family, extracting genomic DNA in peripheral Blood leucocyte by QIAmp Blood kit (Qiagen, Hilden, Germany), and measuring the concentration and purity of DNA by using a Qubit Fluorometer and agarose gel electrophoresis, wherein the obtained genomic DNA OD260/OD280 of each sample is between 1.7 and 2.0, the concentration is not less than 50 ng/microliter, and the total amount is not less than 3 micrograms.

3. Exon capture sequencing

The inventor utilizes a NimbleGen SeqCap EZ Human exon Library v3.0 whole exon capture platform and combines the high-throughput sequencing technology of Illumina Hiseq2000 to sequence the exon sequences of two patients in the family of the autosomal recessive non-integrated deafness patients and parents with normal phenotype.

The method comprises the following specific steps:

1) each genomic DNA sample was randomly fragmented into fragments of about 250-300bp using a sonicator (Covaris 2, Massachusetts, USA), and then adaptor-prepared libraries were ligated to both ends of the fragments according to the manufacturer's instructions (see: http:// www.illumina.com/Illumina/Solexa Standard library Specification, provided, incorporated herein by reference in its entirety).

2) The Library is purified, hybridized and enriched with a capture reagent Biotinylated DNA Library through linear amplification of Ligation-mediated PCR (LM-PCR), and subjected to LM-PCR linear amplification, and the Library can be subjected to on-machine sequencing after qualified detection, so that original sequencing data can be obtained. Wherein the sequencing platform is Illumina Hiseq2000, the reading length is 90bp, and the average sequencing depth of each sample is at least 50 x.

3) Mutation detection, annotation, and database comparison

The raw sequencing data obtained above was processed using Illumina based Software 1.7 and, after filtering for decontamination, aligned to a reference genome UCBI NCBI37/hg19 using SOAPaligner/SOAP2 (see: Li R, Li Y, Kristiansen K, et al, SOAP: short oligonucleotide alignment program. bioinformatics 2008,24(5): 713-714; Li R, Yu C, Li Y, ea al, SOAP2: an improved assay tool for short alignment. bioinformatics 2009,25(15):1966-1967, which is incorporated herein by reference in its entirety) to obtain a unique alignment sequence aligned to the genome. The genotype of the target region is then determined using SOAPsnp (see: Li R, Li Y, Fang X, YangH, et al, SNP detection for mapping parallel gene re-ordering. genome Res 2009,19(6):1124-1132, which is incorporated herein by reference in its entirety).

As a result, 94316 Single Nucleotide Polymorphisms (SNPs) and 6774 insertions/deletions (Indels) were found in case 1(II1), 93434 SNPs and 6807 Indels were found in case 2(II:2), 93657 SNPs and 6753 Indels were found in the father (I:1), and 99942 SNPs and 7055 Indels were found in mother (I: 2). Then, filtering public databases such as dbSNP database (http:// hgdownloadload. cse. ucsc. edu/goldenPath/hg19/database/snp132.txt. gz), HapMap database (ftp:// ftp. ncbi. nlm. nih. gov/HapMap), thousand genome database (ftp:// ftp. 1000genome. ebi. ac. uk/1/ftp), yellow database (http:// yh. genomics. org. cn /) and the like, all known variations (variation values are high, common polymorphisms that are usually not pathogenic MAF) with the allele frequency of more than 0.005 in the database are removed, and functional prediction of SNPs is performed by using SIFT software.

The sensorineural deafness belongs to two genetic modes of frequently recessive and frequently obvious, and the inventor uses recessive genetic strategy analysis by combining the actual conditions of families. And (3) taking intersection of the analyzed information adding result and a gene list related to deafness reported at present, and verifying to obtain 22 complex heterozygous mutant genes and two homozygous mutant genes. The inventor synthesizes sequencing quality and information analysis data screening reference, and combines recessive genetic patterns: namely, parental heterozygous mutation, child homozygous mutation or parental same gene with one mutation and two mutations simultaneously owned by child (compound heterozygous), the mutation on the ARNSHL known gene PTPRQ is found to be qualified. Two missense mutations on PTPRQ are common to patients: c.3125A > G (p.D1042G), c.5981A > G (p.E1994G). According to informatics analysis, two missense mutations in PTPRQ can form a compound heterozygous mutation, as parents carry one mutation in this family.

PTPRQ is a known deafness causing gene, which comprises 45 exons and encodes 260.924KDa protein. The protein encoded by PTPRQ belongs to the family of type III receptor protein tyrosine phosphatases (ptpases), which function to catalyze phosphorylation of phospho-and phosphoinositides during cell proliferation and differentiation. Research shows that the mutation of the gene can cause autosomal recessive hereditary hearing loss.

Furthermore, sanger sequencing verification is carried out on two patients and normal parents of the two patients, and the result shows that the mutation sites accord with cosegregation of an autosomal recessive inheritance pattern. Thus, the inventors considered that the c.3125A > G (p.D1042G) and c.5981A > G (p.E1994G) mutations of the PTPRQ gene may be highly likely to be novel pathogenic mutations of autosomal recessive non-synthetic deafness.

Example 2 sequencing validation by Sanger method

All family members (including 2 patients and 2 normal family members) in the family of autosomal recessive non-syngeneic deafness patients described in example 1 and 328 randomly picked out family-of-normal persons were tested for PTPRQ genes, respectively: designing primers aiming at c.3125A > G and c.5981A > G mutation of the PTPRQ gene, obtaining related sequences of mutation sites by PCR amplification, product purification and sequencing, and verifying the correlation between the c.3125A > G and c.5981A > G mutation of the PTPRQ gene and autosomal recessive and non-synthetic deafness according to whether the sequence determination result belongs to a mutant type or a wild type.

The method comprises the following specific steps:

1. DNA extraction

According to the method for extracting DNA described in example 1, genomic DNA in peripheral venous blood of the prepared subjects was extracted and prepared, respectively, for use.

2. Primer design and PCR reaction

Firstly, referring to a human genome sequence database GRCh37/hg19, exon-specific primers aiming at c.3125A > G and c.5981A > G mutations of the PTPRQ gene are designed, and the specific sequences are as follows:

mutations	Upstream primer (5 '→ 3', SEQ ID NO:)	Downstream primer (5 '→ 3', SEQ ID NO:)
			c.3125A>G	TGTCGATTTTCCTAAAACAACAT(3)	GCAGTTTTCTTGAACAGAAGAGG(4)
c.5981A>G	TGATTTTGGGGATGTCCATT(5)	TCAAGGCCAGAGTCCTTCAT(6)

Then, PCR reaction systems of the genome DNA samples are prepared and PCR reaction is carried out according to the following mixture ratio:

reaction system (20 μ l):

and (3) PCR reaction conditions:

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

3. Sequencing

Directly carrying out DNA sequencing on the PCR amplification product of the genome DNA sample of each subject obtained in the step 2. Wherein, sequencing is carried out by an ABI3730 sequencer.

Based on sequencing results, mutation investigation was performed on c.3125A > G (p.D1042G) and c.5981A > G (p.E1994G) mutation sites of PTPRQ gene in the family of autosomal recessive non-synthetic deafness patients of the present invention, and as a result, it was found that both patients in the family carried complex heterozygous mutations of c.3125A > G (p.D1042G) and c.5981A > G (p.E1994G), while parents who exhibited normal carried one of the heterozygous mutations: the father carries only missense mutations of c.5981A > G (p.E1994G), while the mother carries only missense mutations of c.3125A > G (p.D1042G) (see FIG. 4 for specific results). Furthermore, of the 328 normal control samples, only 2 samples had the c.3125a > G mutation, while none of the c.5981a > G mutations were present in these samples. Wherein, FIG. 4 is a representative Sanger sequencing verification peak diagram of PTPRQ gene c.3125A > G and c.5981A > G mutation sites of all family members in the ARNSHL patient family.

Thus, it is further proved that c.3125A > G (p.D1042G) and c.5981A > G (p.E1994G) of the PTPRQ gene are new pathogenic sites of autosomal recessive nonsynthetic deafness, and complex hybrid mutation of c.3125A > G and c.5981A > G of the PTPRQ gene can cause the disease.

Example 3 detection kit

Preparing a detection kit comprising primers capable of detecting c.3125a > G and c.5981a > G mutations of PTPRQ gene for screening biological samples susceptible to autosomal recessive nonsynthetic deafness, wherein the primers are specific primers for exons of PTPRQ gene, the sequences of which are as shown in example 2 SEQ ID NO: 3-6.

The method for screening the biological sample susceptible to autosomal recessive nonsynthetic deafness by using the kit comprises the following specific steps: extracting DNA of a person to be tested according to the method of the step 2 in the example 1, carrying out PCR reaction by taking the extracted DNA as a template and the exon-specific primers of the PTPRQ gene (the PCR reaction system and the reaction conditions are shown in the example 2), purifying PCR products according to a conventional method in the field, sequencing the purified products, and observing whether the sequence obtained by sequencing has c.3125A > G and c.5981A > G mutations at the same time, so that whether two PTPRQ gene mutants exist in the DNA of the person to be tested at the same time can be effectively detected, and whether the person to be tested is susceptible to autosomal recessive nonsynthetic deafness can be effectively detected, and further, a biological sample susceptible to autosomal nonsynthetic deafness can be screened from the person to be tested.

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, materials, or characteristics 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.

Claims (10)

1. An isolated nucleic acid encoding a PTPRQ mutant, which is substantially identical to SEQ ID NO: 1, or a c.5981A > G mutation,

optionally, the nucleic acid is DNA.

2. An isolated polypeptide that hybridizes to SEQ ID NO: 2, or the p.E1994G mutation,

optionally, the polypeptide is encoded by the nucleic acid of claim 1.

3. A system for screening a biological sample susceptible to autosomal recessive non-synthetic deafness, comprising:

a nucleic acid extraction device for extracting a nucleic acid sample from the biological sample;

a nucleic acid sequence determining device connected with the nucleic acid extracting device and used for analyzing the nucleic acid sample so as to determine the nucleic acid sequence of the nucleic acid sample;

a judging means connected to the nucleic acid sequence determining means so as to compare the nucleic acid sequence of the nucleic acid sample or a complementary sequence thereof with the nucleic acid sequence of SEQ ID NO: 1, and judging whether the biological sample is susceptible to autosomal recessive nonsynthetic deafness or not by judging whether the biological sample has compound heterozygous mutation of c.3125A > G and c.5981A > G.

4. The system of claim 3, wherein the nucleic acid extraction device further comprises:

an RNA extraction unit for extracting an RNA sample from the biological sample; and

a reverse transcription unit connected to the RNA extraction unit for performing a reverse transcription reaction on the RNA sample to obtain a cDNA sample, the cDNA sample constituting the nucleic acid sample.

5. The system of claim 3, wherein the nucleic acid sequence determination device further comprises:

a library construction unit for constructing a nucleic acid sequencing library for the nucleic acid sample; and

and the sequencing unit is connected with the library construction unit and used for sequencing the nucleic acid sequencing library so as to obtain a sequencing result consisting of a plurality of sequencing data.

6. The system of claim 5, wherein the library construction unit further comprises:

a PCR amplification module, wherein a PTPRQ gene exon specific primer is arranged in the PCR amplification module so as to perform PCR amplification on the nucleic acid sample by using the specific primer,

optionally, the step of (a) is carried out,

the PTPRQ gene exon-specific primers have the sequence shown in SEQ ID NO: 3-4;

the PTPRQ gene exon specific primer has the nucleotide sequence shown as SEQ ID NO: 5-6 of the sequence of nucleotides shown in the sequence table,

optionally, the sequencing unit comprises at least one selected from the group consisting of hipseq 2000, SOLiD, 454 and a single molecule sequencing device.

7. A kit for screening a biological sample susceptible to autosomal recessive non-integrated deafness, comprising:

suitable for detecting a peptide corresponding to SEQ ID NO: 1, and a PTPRQ gene mutant having a c.3125a > G mutation compared to SEQ ID NO: 1 in comparison with a reagent for a mutant PTPRQ gene having a c.5981A > G mutation,

optionally, the reagent is a nucleic acid probe or primer,

optionally, the polypeptide is directed against a polypeptide that is identical to SEQ ID NO: 1, and the nucleic acid probe or primer has the nucleotide sequence shown as SEQ ID NO: 3-4;

for sequences similar to SEQ ID NO: 1, and the nucleic acid probe or primer has the nucleotide sequence shown in SEQ ID NO: 5-6.

8. A construct comprising the isolated nucleic acid encoding a PTPRQ mutant of claim 1.

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

10. A method of constructing a drug screening model, comprising:

allowing at least a portion of the cells of the animal to simultaneously express an amino acid sequence identical to SEQ ID NO: 1, and a nucleic acid having a c.3125a > G mutation as compared to SEQ ID NO: 1 in comparison to a nucleic acid having a c.5981A > G mutation,

optionally, the animal is a mouse, pig, dog, primate.