CN102304584A - GJB2 gene p.V37I mutation diagnostic kit for diagnosing late-onset deafness - Google Patents
GJB2 gene p.V37I mutation diagnostic kit for diagnosing late-onset deafness Download PDFInfo
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- CN102304584A CN102304584A CN201110273426A CN201110273426A CN102304584A CN 102304584 A CN102304584 A CN 102304584A CN 201110273426 A CN201110273426 A CN 201110273426A CN 201110273426 A CN201110273426 A CN 201110273426A CN 102304584 A CN102304584 A CN 102304584A
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Abstract
The invention relates to application of GJB2 gene p.V37I mutation to the preparation of products for diagnosing late-onset deafness. The GJB2 gene p.V37I mutation is p.V37I exclusive mutation. The invention also provides a GJB2 gene p.V37I mutation diagnostic kit for diagnosing the late-onset deafness and application of the kit. The GJB2 gene p.V37I mutation diagnostic kit has the advantages that: a common molecular identifier, namely the GJB2 gene p.V37I exclusive mutation related to the late-onset deafness highly is discovered at home and abroad for the first time; a molecular identifier-based gene screening method and the preferential application range of the molecular identifier in the screening of neonates are determined; and the alarm time of the late-onset deafness caused by the GJB2 gene p.V37I exclusive mutation is advanced to a neonate period, and the GJB2 gene p.V37I mutation diagnostic kit is widely applied to the early detection, intervention and rehabilitation of genetic susceptible late-onset deafness, the screening and diagnosis of deafness genes, late-onset deafness hearing-gene combined screening and other relevant study fields.
Description
Technical field
The present invention relates to a kind of test kit, specifically, is a kind of GJB2 gene p.V37I sudden change diagnostic kit that is used to diagnose tardy induced deafness.
Background technology
Hearing is one of indispensable factor in the development of speech process.(Permanent Childhood Hearing Impairment, infant PCHI) carry out early stage prevention and intervene and can obtain good speech and cognitive development to suffering from the permanent hearing loss of children.At present; Ubiquity UNHS (Universal Newborn Hearing Screening; UNHS) but the neonatal hearing loss of early discovery; But for the tardy induced deafness (sickness rate was about 0.25 ‰-0.99 ‰ among the children at 9 years old) that betides after neonatal period; Owing to lack effective risk factors assessment and EARLY RECOGNITION sign, still lack effective early discovery means.
The UNHS technology is extensively carried out in countries in the world, can and carry out early intervention through this examination early discovery neonatal period hearing loss, with dysplasias such as the speech of avoiding newborn deaf youngster to be brought by dysaudia, language.But the tardy induced deafness that betides after neonatal period can not be found by UNHS, and still lacking at present can early warning or the effective means of early discovery.The deaf initial discovery of delayed mainly relies on the head of a family that the observation or the patient oneself of infant hearing situation are detected at present, and discovery time is later, influences the normal development of learning and communication abilities such as speech, language easily because of hearing loss.
The deaf gene detection technique at home and abroad was used widely over past ten years, can effectively detect and diagnosed hereditary hearing impairment.Can carry out early warning to the infant that has tardy induced deafness genetic risk on the tardy induced deafness gene test principle, in conjunction with the hearing examination to reach the purpose of early detection, intervention and rehabilitation.But the bottleneck of this method is still not know the clear and definite early molecule sign (transgenation) that causes common tardy induced deafness genetic predisposition at present, therefore can't carry out tardy induced deafness gene test targetedly.
The GJB2 gene is the common deaf ospc gene of Chinese population, and its transgenation can cause the recessive inheritance induced deafness.In the numerous sudden changes of GJB2 gene, p.V37I sudden change is comparatively common, and the individuality that has p.V37I exclusiveness sudden change (comprise that p.V37I isozygotys, and two kinds of genotype of compound heterozygosis of p.V37I and the sudden change of another one afunction) is prone to light moderate deafness.
Chinese patent document CN101363054A discloses a kind of method of vitro detection hereditary hearing impairment hearing loss relative connexin 26 gene GJB2 sudden change and has detected and use test kit; Through carrying out polymerase chain reaction; Complete amplification is carried out in GJB2 gene basis promoter region, exons 1, exon 2 and shear zone; Carry out determined dna sequence then; Detect the existence of GJB2 transgenation, can improve the recall rate of GJB2 transgenation and the diagnosis of GJB2 gene-correlation sex-controlled inheritance induced deafness.Chinese patent document CN1873027A discloses a kind of primer and application thereof that is used for in-vitro diagnosis non-syndrome autosomal recessive inheritance GJB 2 mutation of deaf gene; Be provided for the primer of full code area sudden change of in-vitro diagnosis non-syndrome autosomal recessive inheritance deaf gene GJB2 gene and the sudden change of 233-235delC focus; Amplification that can the important shearing sequence in full code area of disposable completion GJB2 and both sides; This invention also provides kit or the similar products that contain this primer and ApaI restriction endonuclease, is applicable to the non-syndrome autosomal recessive inheritance deaf gene GJB2 large-scale examination of gene mutation carrying out and diagnosis, genetic counselling.Li Jing etc. utilization PCR directly the method for order-checking the NSHL patient of 139 routine affinity-less relations is carried out Mutation Screening; Two kinds of sudden change p.F115C and p.V37I are building up to the pEGFP expression vector; And transfection Hela cell, study its cell expressing and location situation and (see for details: non-syndrome induced deafness (NSHL) patient
Cx26The Subcellular Localization of gene mutation analysis and two kinds of mutant. heredity, 2009,31 (7): 705-712.).But international, domestic still haveing nothing to do in sudden change of p.V37I exclusiveness and the tardy induced deafness report that is mutually related.
Summary of the invention
The objective of the invention is to deficiency of the prior art, a kind of application of GJB2 gene p.V37I sudden change is provided.
One purpose more of the present invention is that a kind of GJB2 gene p.V37I sudden change diagnostic kit that is used to diagnose tardy induced deafness is provided.
Another purpose of the present invention is, a kind of application that is used to diagnose the GJB2 gene p.V37I sudden change diagnostic kit of tardy induced deafness is provided.
For realizing above-mentioned purpose, the technical scheme that the present invention takes is: the application of a kind of GJB2 gene p.V37I sudden change in the tardy induced deafness product of preparation diagnosis, described p.V37I sudden change is the sudden change of p.V37I exclusiveness.
Described p.V37I exclusiveness sudden change is the sudden change of p.V37I homozygous genotype.
Described p.V37I exclusiveness sudden change is the compound heterozygous genes type sudden change of a p.V37I and an afunction sudden change.
For realizing above-mentioned second purpose, the technical scheme that the present invention takes is: a kind of GJB2 gene p.V37I sudden change diagnostic kit that is used to diagnose tardy induced deafness, and described test kit contains two pairs of nest-type PRC amplimers, and described amplimer is:
The primer of first round PCR: GJB2 FO (SEQ ID NO.3) and GJB2 RO (SEQ ID NO.4); Second primer of taking turns PCR: GJB2 FI (SEQ ID NO.5) and GJB2 RI (SEQ ID NO.6).
Described test kit also comprises dNTP, archaeal dna polymerase and damping fluid thereof.
For realizing above-mentioned the 3rd purpose, the technical scheme that the present invention takes is: the application of described test kit in the deaf product of preparation diagnosis delayed.
The invention has the advantages that:
1, the present invention identifies with the common molecule of tardy induced deafness height correlation in international, the domestic discovery first:
GJB2The sudden change of gene p.V37I exclusiveness, and established based on the gene screening method of this molecule sign and the priority application scope in neonatal screening thereof;
2, through discovery and related application Journal of Sex Research to the common genetic predisposition early molecule of tardy induced deafness sign, can with by
GJB2The gene p.V37I exclusiveness caused tardy induced deafness pre-warning time that suddenlys change is advanced to neonatal period; Can be widely used in the tardy induced deafness early detection of genetic predisposition, intervention and rehabilitation, deaf ospc gene examination and diagnosis, tardy induced deafness hearing-gene associating examination and Related Research Domain.
Description of drawings
Accompanying drawing 1 is a nest-type PRC amplification synoptic diagram.
Accompanying drawing 2 is
GJB2The condition of all exon pcr amplifications of gene.
Accompanying drawing 3 is nest-type PRC amplified production electrophoretic effects figure.
Accompanying drawing 4 is
GJB2Gene p.V37I homozygous mutation.
Accompanying drawing 5 with accompanying drawing 6 is
GJB2The sudden change of gene p.V37I exclusiveness.
Embodiment
Below in conjunction with accompanying drawing embodiment provided by the invention is elaborated.
GJB2Gene is the common deaf ospc gene of Chinese population, and its transgenation can cause the recessive inheritance induced deafness.
GJB2In the numerous sudden changes of gene, p.V37I sudden change is comparatively common, and the individuality that has p.V37I exclusiveness sudden change (comprise that p.V37I isozygotys, and two kinds of genotype of compound heterozygosis of p.V37I and the sudden change of another one afunction) is prone to light moderate deafness.Described
GJB2The aminoacid sequence of gene is shown in SEQ ID NO.1, and the 37th of described p.V37I sudden change expression GJB2 aminoacid sequence sports Isoleucine (I) by Xie Ansuan (V), sees SEQ ID NO.2.
P.V37I exclusiveness sudden change comprises that p.V37I isozygotys (a pair of gene of somatocyte all carries the p.V37I sudden change), and the compound heterozygosis of p.V37I and the sudden change of another one afunction (one of a pair of gene of somatocyte carries p.V37I and suddenlys change, and another carries and can cause
GJB2Other sudden change of gene function forfeiture) two kinds of genotype, these genotype characteristics have function for all
GJB2Gene all has the p.V37I sudden change, i.e. p.V37I exclusiveness sudden change.
Through surpassing the research of 1000 routine samples, the present invention finds that at first the carrying rate of p.V37I in Chinese population is 6.1% (n=3032 sees table 1), is the modal deaf gene mutational site of being found at present.Previously research shows, when individuality carries
GJB2During the sudden change of gene p.V37I exclusiveness, its possibility that produces light moderate auditory dysesthesia increases greatly, and some cases shows as carrying out property auditory dysesthesia.In the research process of the present invention, suppose and proved
GJB2Gene p.V37I exclusiveness sudden change is relevant with tardy induced deafness, can be used as early molecule and identifies and carry out tardy induced deafness risk assessment and gene early warning, and process is following:
Through outpatient service and the 21427 routine Shanghai area children of kindergarten examinations, collect the tardy induced deafness case of 45 example Chinese populations suspicious or that make a definite diagnosis altogether.Bilateral ear UNHS passed through when confirmed cases were defined as birth; Before 9 years old, developed into the tardy induced deafness of bilateral (the better ear PTA of hearing is at 0.5k, 1k, 2k and the average threshold of audibility of 4 kHz >=40 dBHL); (n=14, age 2-9 year, average age of onset 5.0 years old); Non-confirmed cases group is defined as when birth and goes UNHS, at 5 years old or occur bilateral PCHI (n=31, average age of onset 8 years old, age 5-12 year) later on.The GJB2 detection in Gene Mutation detects through pcr amplification and two-way order-checking.
GJB2The sudden change of gene p.V37I exclusiveness is 21.4% (3/14) in definite case group incidence, and non-confirmed cases group incidence is 19.4% (6/14), and in control group, incidence is 0.4% (6/1516) among the 1516 routine newborn infants, sees following table 1 for details.
The carrying rate of table 1 p.V37I exclusiveness sudden change in each crowd
| ? | Amount to | P.V37I exclusiveness sudden change (%) | PValue * |
| The contrast neonate group | 1516 | 6 (0.40) | - |
| PCHI makes a |
14 | 3 (21.4) | 9.0×10 -5§ |
| The non-group of making a definite diagnosis of PCHI | 31 | 6 (19.4) | 9.8×10 -8 § |
| PCHI merging group | 45 | 9 (20.0) | 1.4×10 -10 § |
| Primary dcreening operation passes through neonate group | 1405 | 2 (0.14) | - |
| Multiple sieve passes through neonate group | 99 | 2 (2.0) | 0.024 §§ |
| The hearing diagnosis of changing the place of examination is a hearing normal newborn group | 173 | 10 (5.8) | 1.7×10 -8 ? §§ |
*Through the definite stochastic method statistics of Fisher, two-tailed test;
§Compare with the contrast neonate group;
§ §Compare through neonate group with primary dcreening operation.
This group data shows
GJB2The sudden change of gene p.V37I exclusiveness (is used Fisher ' s exact test statistical method, is made a definite diagnosis group with tardy induced deafness is closely related
P=9.0 * 10
-5, the non-group of making a definite diagnosis
P=9.8 * 10
-8Tardy induced deafness group after the two merges
P=1.4 * 10
-10, all difference is remarkable), be the important common mutations of Chinese tardy property PCHI crowd, account for sum 20%.Above result proves
GJB2Gene p.V37I exclusiveness sudden change is closely related with tardy induced deafness, can be used as early molecule and identifies and carry out tardy induced deafness risk assessment and gene early warning.
The present invention has further established the priority application scope of p.V37I gene screening in the newborn infant through research: use otoacoustic emission (DPOAE) method to carry out among the newborn infant of UNHS, have at least the incidence of once not suddenling change through p.V37I exclusiveness among the newborn infant of hearing examination significantly to raise: its incidence is 0.14% (2/1405) among the newborn infant that primary dcreening operation passes through; Primary dcreening operation does not pass through, but among the newborn infant who passes through when sieving again its incidence be 2.0% (2/99,
P=0.024); And primary dcreening operation sieves again and all do not pass through, but be diagnosed as when carrying out hearing diagnosis among the normal newborn infant of hearing its incidence be 5.8% (10/173,
P=1.7 * 10
-8) (seeing table 1).This group data support the sudden change of p.V37I exclusiveness can cause connatae subclinical hearing loss from another point of view, and the risk that tardy induced deafness takes place its carrier obviously raises.
Based on this research, it is considered herein that this tardy induced deafness genetic predisposition molecule sign
GJB2The discovery of gene p.V37I exclusiveness sudden change has important clinical meaning and application prospect: in having at least once not through the newborn infant crowd of UNHS, carry out the sudden change of p.V37I exclusiveness and detect, the children that carry this sudden change are carried out effective audiology tracking monitor can find and intervene tardy induced deafness early.The risk crowd that do not carry that tardy induced deafness takes place the individuality that carries p.V37I exclusiveness sudden change significantly raises; The high risk population that should be classified as the tardy induced deafness line period property audiology of going forward side by side is followed up a case by regular visits to, with the tardy induced deafness of early discovery and avoid serious consequences such as speech dysplasia.
Embodiment 2 detection methods
Key instrument equipment is seen table 2.
Table 2 key instrument equipment
| The instrument title | Production company or model |
| PCR appearance: ABI2720 Thermal cycler | U.S. Applied Biosystems company |
| Pure water appearance: Millipore Direct Q3 | Germany Millipore company |
| Sequenator: ABI Prism 3730 | U.S. Applied Biosystems company |
| The detection of nucleic acids appearance | Thermo NANODROP 2000 |
| Refrigerated |
Thermo LEGEND RT+ |
| Refrigerated centrifuge 2 | Thermo LEGEND MICRO 17R |
| Ice-making machine | Scotman AF100 |
| The fully automatic digital gel imaging system | Tanon 3500 |
| Electronic scales FA2004 | The permanent flat scientific instrument company limited of Shanghai wink space |
| Electrophoresis apparatus | The horizontal HE120 of agarose, HE90 |
| Constant water bath box: DK-8AX | Shanghai technology company limited of permanent section |
| 8 roads trace liquid feeding rifle (0.1ml-10ml) | Eppendorf company |
| 8 roads trace liquid feeding rifle (1ml-100ml) | Eppendorf company |
| 0.1ml-2.5ml micro-liquid feeding rifle | Eppendorf company |
| 2ml-20ml trace liquid feeding rifle | Eppendorf company |
| 20ml-200ml trace liquid feeding rifle | Eppendorf company |
| 100ml-1000ml trace liquid feeding rifle | Eppendorf company |
| 96 hole PCR micro-reaction plates (overskirt limit) | Match brilliant bio tech ltd |
One, DNA extraction
The centrifugal column type poba gene group DNA extraction test kit (TIANamp Blood DNA Kit) that adopts TIANGEN Biotech (Beijing) Co., Ltd. to produce extracts the complete genome DNA in the venous blood.Reagent is formed following table (table 3):
The moiety of table 3 DNA extraction test kit
| Test kit is formed | 50 times/box |
| Cell pyrolysis liquid | 60ml |
| |
15 ml |
| |
15 ml |
| |
13 ml |
| Rinsing |
15 ml |
| |
15 ml |
| Proteinase K | 1 |
| Adsorption column CB3 | 50 |
| Collection tube (2ml) | 50 |
| 1.5ml aseptic centrifuge tube | 50 |
| Conservation condition | Room temperature (15-25 ℃) is preserved |
Step is following:
1. blood sampling 600 μ l are in the aseptic centrifuge tube of 2ml; The cell pyrolysis liquid CL that adds 1200 μ l; Put upside down mixing, the centrifugal 1min of 10000rpm inhales and removes supernatant; Stay the nucleus deposition (if lysis is thorough; Then managing the end is white precipitate, if then cracking is not thorough for redness, can repeat above step once); In the centrifugal nucleus deposition of collecting, add 200 μ l damping fluid GS, vibration is to thorough mixing.
2. add 20 μ l Proteinase K solution, mixing.
3. add 200 μ l damping fluid GB, fully put upside down mixing, 56 ℃ of water-bath 10min, during put upside down mixing.
4. add 200 μ l damping fluid GB, fully put upside down mixing, place 10min for 56 ℃, put upside down mixing therebetween for several times, the solution strain is limpid.
5. add 200 μ l dehydrated alcohols, fully put upside down mixing, flocks may appear in this moment.
6. will go up step gained solution and flocks and all add among the adsorption column CB3, the centrifugal 30s of 12000rpm outwells the waste liquid in the collection tube, and CB3 puts into collection tube with adsorption column.
7. in adsorption column CB3, add 500 μ l damping fluid GD, the centrifugal 30s of 12000rpm outwells the waste liquid in the collection tube, and CB3 puts into collection tube with adsorption column.
8. in adsorption column CB3, add 700 μ l rinsing liquid PW, the centrifugal 30s of 12000rpm outwells the waste liquid in the collection tube, and CB3 puts into collection tube with adsorption column.
9. in adsorption column CB3, add 500 μ l rinsing liquid PW, the centrifugal 30s of 12000rpm outwells the waste liquid in the collection tube.
10. adsorption column CB3 is put back in the collection tube, the centrifugal 2min of 12000rpm outwells waste liquid.Place room temperature to place several minutes adsorption column CB3, thoroughly to dry rinsing liquid remaining in the sorbing material.
11. adsorption column CB3 is changed in the clean centrifuge tube, and to the unsettled dropping 100 μ l elution buffer TB in adsorption film mid-way, room temperature is placed 5min, the centrifugal 2min of 12000rpm collects solution in the centrifuge tube, i.e. DNA stoste.
12. after utilizing detection of nucleic acids appearance (NANODROP 2000) that DNA stoste is carried out quantitative and purity check, get the working concentration that a certain amount of DNA stoste is diluted to 10ng/ μ l ,-20 ℃ of preservations are subsequent use, DNA stoste-80 ℃ preservation.
Two, nest-type PRC amplifying target genes fragment
Nest-type PRC (Nest PCR); Promptly utilize two cover primers that each target gene fragment that need increase is carried out the twice PCR amplification; A pair of primer away from target gene fragment is an outside primer; It is the primer of first round PCR; A pair of primer near target gene fragment is inboard primer, i.e. second primer of taking turns PCR.Synoptic diagram is seen accompanying drawing 1, and accompanying drawing 1 is a nest-type PRC amplification synoptic diagram.
GJB2The two-wheeled primer of gene is following:
GJB2?FO:?TGGTGTTTGCTCAGGAAGAG?（SEQ?ID?NO.3）
GJB2?RO:?TGTGGCATCTGGAGTTTCAC?（SEQ?ID?NO.4）
GJB2?FI:?ATGCTTGCTTACCCAGACTC?（SEQ?ID?NO.5）
GJB2?RI:?TTGGGAAATGCTAGCGACTG?（SEQ?ID?NO.6）
Because the nest-type PRC reaction has the twice PCR amplification; Thereby the possibility (all the complementary primer is seldom because with two cover primers) that has reduced a plurality of target sites that increase has increased the susceptibility and the specificity that detect, therefore also pollutes easily (negative control is set can be avoided).
Pcr amplification reagent sees the following form 4.
Each composition relevant information in the table 4 pcr amplification system
| Reagent | Concentration | Preservation condition | Manufacturer |
| Distilled water (ddH 2O） | — | Room temperature | Millipore system water appearance |
| DNTP (comprising dATP, dTTP, dGTP, dCTP) | 2.5mmol/μl | -20℃ | It root biochemical technology company limited |
| Taq DNA Polymerase | 2.5U/μl | -20℃ | It root biochemical technology company limited |
| Taq Buffer（Mg 2+ free） | 10X | -20℃ | It root biochemical technology company limited |
| Mg 2+ | 2.5mmol/μl | -20℃ | It root biochemical technology company limited |
| Pcr amplification primer (being diluted to working concentration) | 5μmol/μl | -20℃ | It is synthetic that worker biotech firm is given birth in Shanghai |
GJB2The reaction system that all exon first round of gene and second are taken turns pcr amplification all is 25 μ l, comprises 10ng/ μ l genomic dna 2 μ l when taking turns PCR (second added be the product of the first round PCR of 2 μ l), 10 * damping fluid (Mg
2+Free) 2.5 μ l, 2.5mM dNTP 2 μ l, 2.5mM Mg2+ 1.5 μ l, each 1 μ l of the forward and reverse primer of 5 μ M, archaeal dna polymerase 1.0 U add distilled water and mend to 25 μ l, specifically see the following form 5.
Table 5
GJB2The reaction system of each exon pcr amplification of gene
| The composition title | Components and concentration | The composition volume |
| ddH 2O | ? | 15.6μl |
| Taq Buffer | 10X | 2.5μl |
| dNTP | 2.5mmol/μl | 2μl |
| Mg 2+ | 2.5mmol/μl | 1.5μl |
| Forward primer | 5μmol/μl | 1μl |
| Reverse primer | 5μmol/μl | 1μl |
| Template DNA | 10ng/μl | 1μl |
| Taq DNA Polymerase | 2.5U/μl | 0.4μl |
| Amount to | ? | 25μl |
Be reflected on the ABI2720 thermal cycler and accomplish,
GJB2It is identical that the first round of gene and second is taken turns the pcr amplification condition: behind 94 ℃ of preparatory sex change 1min, 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 33 circulations of 30s, after reaction finishes again 72 ℃ extend 10min, accompanying drawing 2 is seen in 4 ℃ of preservations, accompanying drawing 2 is
GJB2The condition of all exon pcr amplifications of gene.
Three, the PCR product is identified
Glue: take by weighing 0.75g agar Icing Sugar; Add and to be mixed with 1.5% agarose in the 50mlTAE electrophoretic buffer; Behind the vibration mixing; Microwave heating 1min; Be cooled to and add 1 μ lEB (ethidium bromide) dyestuff about 60 ℃; Pour into behind the mixing in the glue bed that assembles, 30min treats carefully to extract after agarose solidifies fully application of sample comb to get glue subsequent use under the room temperature.
Application of sample: 1 μ l Loading Buffer (tetrabromophenol sulfonphthalein) takes turns the PCR product with 4 μ l second and mixes, and careful the adding in the well after micropipet is blown and beaten evenly, 4 μ l DL2000 are as DNA standard molecular weight (Marker) while application of sample.Whether each application of sample all adds negative control (not adding dna profiling during first round PCR) simultaneously contaminated to judge product.
Electrophoresis: will go up excellent gel in electrophoresis apparatus, and carry out the constant voltage electrophoresis with 90V voltage.Electrophoresis time is 50min.
Scanning: electrophoresis places the electrophoresis imaging analysis instrument to carry out image scanning gel after finishing, and the result sees accompanying drawing 3, and accompanying drawing 3 is nest-type PRC amplified production electrophoretic effects figure.
Four, interpretation of result
Nest-type PRC second is taken turns product send order-checking company directly to check order, sequencing result is used Sequencher 4.9 softwares analyze.The mutational site is analyzed and is seen accompanying drawing 4-6, and accompanying drawing 4 is
GJB2Gene p.V37I homozygous mutation, accompanying drawing 5 with accompanying drawing 6 is
GJB2The sudden change of gene p.V37I exclusiveness.
More than the research explanation of two aspects, through new molecular marked compound
GJB2Gene p.V37I sudden change carrying out early discovery and the tardy induced deafness case of early warning have the advantage that existing deaf gene detects and the audiology detection does not possess; Can accomplish to detect in early days to tardy induced deafness, early warning and early intervention, avoid the stunted generation of speech.
The present invention has carried out large sample amount gene screening through round pcr and the full gene order surveying method of GJB2 to tardy induced deafness crowd and newborn infant crowd; Identify with the common molecule of tardy induced deafness height correlation in international, the domestic discovery first: the sudden change of GJB2 gene p.V37I exclusiveness, and established based on the gene screening method of this molecule sign and the priority application scope in neonatal screening thereof.
Through
GJB2The detection of gene p.V37I sudden change; But the tardy induced deafness high risk population of early discovery and early warning; Avoid the speech dysplasia of tardy induced deafness case; Reduce social cost and family burden greatly; According to Chinese newborn population 1,600 ten thousand calculating (State Family Planning Commission's data in 2009) in every year; Tardy induced deafness incidence 0.75 ‰; Be to have every year 1.2 ten thousand people that tardy induced deafness takes place; Make these tardy induced deafness infants return normal society need at least 500,000 everyone; Promptly need newly-increased social cost 6,000,000,000 every year, society and family burden are very heavy.
If can carry out early intervention through the early molecule biological diagnosis, can make tardy induced deafness patient deaf and not mute, return normal society, reduce the Financial cost and the psychological burden of society and family greatly.The Application Research of tardy induced deafness molecular marked compound, for tardy induced deafness gene test provides the basis, but the tardy induced deafness case of early discovery more than 20%, the annual social cost that reduces is more than 1,500,000,000.
GJB2Gene p.V37I sudden change is as present unique tardy induced deafness molecular marked compound; Can combine the tardy induced deafness case of gene screening technology early discovery; The discovery and the early warning phase of tardy induced deafness are advanced to neonatal period, thereby make tardy induced deafness patient deaf and not mute, improve deafness and prevent and treat level.Therefore,
GJB2Gene p.V37I sudden change obviously is better than other marker at present as the molecular marked compound of tardy induced deafness.
The present invention is through discovery and related application Journal of Sex Research to the common genetic predisposition early molecule of tardy induced deafness sign, can with by
GJB2The gene p.V37I exclusiveness caused tardy induced deafness pre-warning time that suddenlys change is advanced to neonatal period; Can be widely used in the tardy induced deafness early detection of genetic predisposition, intervention and rehabilitation, deaf ospc gene examination and diagnosis, tardy induced deafness hearing-gene associating examination and Related Research Domain.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; Can also make some improvement and replenish, these improvement and replenish and also should be considered as protection scope of the present invention.
SEQUENCE?LISTING
< 110>Xinhua Hospital Attached to Medical School, Shanghai Jiaotong Univ.
< 120>a kind of GJB2 gene p.V37I sudden change diagnostic kit that is used to diagnose tardy induced deafness
<130> /
<160> 6
<170> PatentIn?version?3.3
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<211> 226
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Met?Asp?Trp?Gly?Thr?Leu?Gln?Thr?Ile?Leu?Gly?Gly?Val?Asn?Lys?His
1 5 10 15
Ser?Thr?Ser?Ile?Gly?Lys?Ile?Trp?Leu?Thr?Val?Leu?Phe?Ile?Phe?Arg
20 25 30
Ile?Met?Ile?Leu?Val?Val?Ala?Ala?Lys?Glu?Val?Trp?Gly?Asp?Glu?Gln
35 40 45
Ala?Asp?Phe?Val?Cys?Asn?Thr?Leu?Gln?Pro?Gly?Cys?Lys?Asn?Val?Cys
50 55 60
Tyr?Asp?His?Tyr?Phe?Pro?Ile?Ser?His?Ile?Arg?Leu?Trp?Ala?Leu?Gln
65 70 75 80
Leu?Ile?Phe?Val?Ser?Thr?Pro?Ala?Leu?Leu?Val?Ala?Met?His?Val?Ala
85 90 95
Tyr?Arg?Arg?His?Glu?Lys?Lys?Arg?Lys?Phe?Ile?Lys?Gly?Glu?Ile?Lys
100 105 110
Ser?Glu?Phe?Lys?Asp?Ile?Glu?Glu?Ile?Lys?Thr?Gln?Lys?Val?Arg?Ile
115 120 125
Glu?Gly?Ser?Leu?Trp?Trp?Thr?Tyr?Thr?Ser?Ser?Ile?Phe?Phe?Arg?Val
130 135 140
Ile?Phe?Glu?Ala?Ala?Phe?Met?Tyr?Val?Phe?Tyr?Val?Met?Tyr?Asp?Gly
145 150 155 160
Phe?Ser?Met?Gln?Arg?Leu?Val?Lys?Cys?Asn?Ala?Trp?Pro?Cys?Pro?Asn
165 170 175
Thr?Val?Asp?Cys?Phe?Val?Ser?Arg?Pro?Thr?Glu?Lys?Thr?Val?Phe?Thr
180 185 190
Val?Phe?Met?Ile?Ala?Val?Ser?Gly?Ile?Cys?Ile?Leu?Leu?Asn?Val?Thr
195 200 205
Glu?Leu?Cys?Tyr?Leu?Leu?Ile?Arg?Tyr?Cys?Ser?Gly?Lys?Ser?Lys?Lys
210 215 220
Pro?Val
225
<210> 2
<211> 226
<212> PRT
< 213>homo sapiens (Homo sapiens)
<400> 2
Met?Asp?Trp?Gly?Thr?Leu?Gln?Thr?Ile?Leu?Gly?Gly?Val?Asn?Lys?His
1 5 10 15
Ser?Thr?Ser?Ile?Gly?Lys?Ile?Trp?Leu?Thr?Val?Leu?Phe?Ile?Phe?Arg
20 25 30
Ile?Met?Ile?Leu?Ile?Val?Ala?Ala?Lys?Glu?Val?Trp?Gly?Asp?Glu?Gln
35 40 45
Ala?Asp?Phe?Val?Cys?Asn?Thr?Leu?Gln?Pro?Gly?Cys?Lys?Asn?Val?Cys
50 55 60
Tyr?Asp?His?Tyr?Phe?Pro?Ile?Ser?His?Ile?Arg?Leu?Trp?Ala?Leu?Gln
65 70 75 80
Leu?Ile?Phe?Val?Ser?Thr?Pro?Ala?Leu?Leu?Val?Ala?Met?His?Val?Ala
85 90 95
Tyr?Arg?Arg?His?Glu?Lys?Lys?Arg?Lys?Phe?Ile?Lys?Gly?Glu?Ile?Lys
100 105 110
Ser?Glu?Phe?Lys?Asp?Ile?Glu?Glu?Ile?Lys?Thr?Gln?Lys?Val?Arg?Ile
115 120 125
Glu?Gly?Ser?Leu?Trp?Trp?Thr?Tyr?Thr?Ser?Ser?Ile?Phe?Phe?Arg?Val
130 135 140
Ile?Phe?Glu?Ala?Ala?Phe?Met?Tyr?Val?Phe?Tyr?Val?Met?Tyr?Asp?Gly
145 150 155 160
Phe?Ser?Met?Gln?Arg?Leu?Val?Lys?Cys?Asn?Ala?Trp?Pro?Cys?Pro?Asn
165 170 175
Thr?Val?Asp?Cys?Phe?Val?Ser?Arg?Pro?Thr?Glu?Lys?Thr?Val?Phe?Thr
180 185 190
Val?Phe?Met?Ile?Ala?Val?Ser?Gly?Ile?Cys?Ile?Leu?Leu?Asn?Val?Thr
195 200 205
Glu?Leu?Cys?Tyr?Leu?Leu?Ile?Arg?Tyr?Cys?Ser?Gly?Lys?Ser?Lys?Lys
210 215 220
Pro?Val
225
<210> 3
<211> 20
<212> DNA
< 213>artificial sequence
<400> 3
tggtgtttgc?tcaggaagag 20
<210> 4
<211> 20
<212> DNA
< 213>artificial sequence
<400> 4
tgtggcatct?ggagtttcac 20
<210> 5
<211> 20
<212> DNA
< 213>artificial sequence
<400> 5
atgcttgctt?acccagactc 20
<210> 6
<211> 20
<212> DNA
< 213>artificial sequence
<400> 6
ttgggaaatg?ctagcgactg 20
Claims (6)
1. the application of GJB2 gene p.V37I sudden change in the tardy induced deafness product of preparation diagnosis is characterized in that described p.V37I sudden change is the sudden change of p.V37I exclusiveness.
2. application according to claim 1 is characterized in that, described p.V37I exclusiveness sudden change is the sudden change of p.V37I homozygous genotype.
3. application according to claim 1 is characterized in that, described p.V37I exclusiveness sudden change is the compound heterozygous genes type sudden change of a p.V37I and an afunction sudden change.
4. a GJB2 gene p.V37I sudden change diagnostic kit that is used to diagnose tardy induced deafness is characterized in that described test kit contains two pairs of nest-type PRC amplimers, and described amplimer is:
The primer of first round PCR: GJB2 FO (SEQ ID NO.3) and GJB2 RO (SEQ ID NO.4); Second primer of taking turns PCR: GJB2 FI (SEQ ID NO.5) and GJB2 RI (SEQ ID NO.6).
5. test kit according to claim 1 is characterized in that described test kit also comprises dNTP, archaeal dna polymerase and damping fluid thereof.
6. according to claim 4 and the application of 5 arbitrary described test kits in the deaf product of preparation diagnosis delayed.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110273426A CN102304584A (en) | 2011-09-15 | 2011-09-15 | GJB2 gene p.V37I mutation diagnostic kit for diagnosing late-onset deafness |
| PCT/CN2011/081409 WO2013037154A1 (en) | 2011-09-15 | 2011-10-27 | Diagnose kit useful for diagnosing p.v37i mutation of gjb2 gene related to tardive deafness |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110273426A CN102304584A (en) | 2011-09-15 | 2011-09-15 | GJB2 gene p.V37I mutation diagnostic kit for diagnosing late-onset deafness |
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| CN102304584A true CN102304584A (en) | 2012-01-04 |
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| CN201110273426A Pending CN102304584A (en) | 2011-09-15 | 2011-09-15 | GJB2 gene p.V37I mutation diagnostic kit for diagnosing late-onset deafness |
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| Country | Link |
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| CN (1) | CN102304584A (en) |
| WO (1) | WO2013037154A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586433A (en) * | 2012-02-14 | 2012-07-18 | 北京科聆金仪生物技术有限公司 | Deafness predisposing gene 12S rRNA (ribosomal ribonucleic acid) 1494C>T fluorescence detection kit and application thereof |
| CN103571846A (en) * | 2012-07-18 | 2014-02-12 | 中国人民解放军总医院 | ATP6V1B2 gene mutant and application thereof |
| CN106906299A (en) * | 2017-04-18 | 2017-06-30 | 吉林省锐吉尔生物科技有限公司 | People's JAK2V617F gene mutation detection kits |
| CN109251979A (en) * | 2018-11-30 | 2019-01-22 | 中国人民解放军第四军医大学 | Phonosensitive nerve deafness Disease-causing gene GJB2 mutation detection kit |
| CN111321218A (en) * | 2020-04-14 | 2020-06-23 | 上海交通大学医学院附属第九人民医院 | Detection kit for GJB2 gene pV37I mutation of late deafness |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101688497A (en) * | 2007-05-28 | 2010-03-31 | 丰田自动车株式会社 | Cetane number estimating device and method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009105543A1 (en) * | 2008-02-19 | 2009-08-27 | Med-El Elektromedizinische Geraete Gmbh | A mutation in the regulatory region of gjb2 mediates neonatal hearing loss within dfnb1 |
| CN101363054B (en) * | 2008-08-18 | 2011-12-21 | 东莞市澳麦尔基因技术有限公司 | Method for detecting hereditary hearing loss relative connexin 26 gene GJB2 mutation and kit for detection |
-
2011
- 2011-09-15 CN CN201110273426A patent/CN102304584A/en active Pending
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101688497A (en) * | 2007-05-28 | 2010-03-31 | 丰田自动车株式会社 | Cetane number estimating device and method |
Non-Patent Citations (5)
| Title |
|---|
| 《AMERICAN JOURNAL OF MEDICAL GENETICS》 20011130 Hao Wu等 Genotype-phenotype correlation of two prevalent GJB2 mutations in chinese newborn infants ascertained from the Universal Newborn Hearing Screening Program 2912-2914 1-6 第152卷, 第11期 * |
| 《中国的遗传学研究--遗传学推动中国西部经济与社会发展--2011年中国遗传学会大会论文摘要汇编》 20110809 李磊等 儿童迟发性耳聋早期基因识别与预警 1-6 , * |
| HAO WU等: "Genotype–phenotype correlation of two prevalent GJB2 mutations in chinese newborn infants ascertained from the Universal Newborn Hearing Screening Program", 《AMERICAN JOURNAL OF MEDICAL GENETICS》, vol. 152, no. 11, 30 November 2001 (2001-11-30), pages 2912 - 2914 * |
| PU DAI等: "GJB2 mutation spectrum in 2063 Chinese patients with nonsyndromic hearing impairment", 《JOURNAL OF TRANSLATIONAL MEDICINE》, vol. 26, no. 7, 14 April 2009 (2009-04-14), pages 1 - 12 * |
| 李磊等: "儿童迟发性耳聋早期基因识别与预警", 《中国的遗传学研究——遗传学推动中国西部经济与社会发展——2011年中国遗传学会大会论文摘要汇编》, 9 August 2011 (2011-08-09) * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586433A (en) * | 2012-02-14 | 2012-07-18 | 北京科聆金仪生物技术有限公司 | Deafness predisposing gene 12S rRNA (ribosomal ribonucleic acid) 1494C>T fluorescence detection kit and application thereof |
| CN102586433B (en) * | 2012-02-14 | 2014-01-29 | 北京科聆金仪生物技术有限公司 | Deafness predisposing gene 12S rRNA (ribosomal ribonucleic acid) 1494C>T fluorescence detection kit and application thereof |
| CN103571846A (en) * | 2012-07-18 | 2014-02-12 | 中国人民解放军总医院 | ATP6V1B2 gene mutant and application thereof |
| CN103571846B (en) * | 2012-07-18 | 2017-07-18 | 中国人民解放军总医院 | ATP6V1B2 gene mutation bodies and its application |
| CN106906299A (en) * | 2017-04-18 | 2017-06-30 | 吉林省锐吉尔生物科技有限公司 | People's JAK2V617F gene mutation detection kits |
| CN109251979A (en) * | 2018-11-30 | 2019-01-22 | 中国人民解放军第四军医大学 | Phonosensitive nerve deafness Disease-causing gene GJB2 mutation detection kit |
| CN111321218A (en) * | 2020-04-14 | 2020-06-23 | 上海交通大学医学院附属第九人民医院 | Detection kit for GJB2 gene pV37I mutation of late deafness |
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|---|---|
| WO2013037154A1 (en) | 2013-03-21 |
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