CN106167800A - EPHA2 genic mutation type and application thereof - Google Patents
EPHA2 genic mutation type and application thereof Download PDFInfo
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Abstract
The open a kind of saltant type EPHA2 gene of the present invention, it has the sequence as shown in SEQ ID NO:1.Invention additionally discloses the polypeptide of saltant type EPHA2 gene code, saltant type EPHA2 gene and/or its coding albumen congenital cataractous early screening, diagnose and/or prepare treatment congenital cataract medicine in purposes, a kind of method of examination congenital cataract biological specimen, the system of a kind of examination congenital cataract biological specimen and the test kit of a kind of examination congenital cataract biological specimen.
Description
Related application
The application asks filing date on May 19th, 2015, entitled " EPHA2 genic mutation type and application thereof "
The priority of Chinese patent application 201510254801.5 and rights and interests, its complete content is incorporated to by referring at this.
Technical field
The present invention relates to biological field, concrete, the present invention relates to abrupt climatic change field, more specifically, the present invention relates to
A kind of saltant type EPHA2 gene, the polypeptide of saltant type EPHA2 gene code, saltant type EPHA2 gene and/or its
The purposes of polypeptide of coding, the method for examination congenital cataract biological specimen, examination congenital cataract biological specimen
System and the test kit of examination congenital cataract biological specimen.
Background technology
Cataract is owing to a variety of causes is the most aging, heredity, locally malnutrition, immunity and Developmental and Metabolic Disorder, wound, in
Poison, radiation etc., cause crystalline lens metabolism disorder, cause crystallins degeneration that muddiness occurs, cataractous patient
Visual deterioration can occur, and serious may be blind.Cataract can be geneogenous, it is also possible to is posteriority, first
It cataract is many to be existed the most at period from prenatal to postnatal, can be broadly divided into anterior polar cataract with hereditary, after the whitest
Cataract, perinuclear cataract and total cataract.
The sickness rate of congenital cataract is about 1-6/10000 people [Apple DJ, Ram J, Foster A, Peng Q (2000)
Elimination of cataract blindness:a global perspective entering the new millenium.Surv
Ophthalmol 45Suppl 1:S1 196.], key factor blind childhood period of being to cause, about 1/3 congenital in vain
Cataract is that inherited genetic factors causes, and its hereditary pattern is mainly autosomal dominant inheritance, AD, also has some recessive inheritances and X
The case of linkage inheritance is in the news [Huang B, He W.Molecular characteristics of inherited congenital
cataracts.Eur J Med Genet 2010；53:347-57.].Cataractous generation may be with lenticular transparency and refraction
The change of rate, nutrition and intercellular communication, cellularity, mobility, shape, the factor such as crystalline lens abnormal development is relevant.
Congenital cataract has higher genetic heterogeneity, and the gene that may result in congenital cataract the most reported is more than 40
Individual, EPHA2 is the gene that one of them existing dominant inheritance pattern has again recessive inheritance's pattern, EPHA2 gene position
In No. 1 the short arm of a chromosome, being made up of 18 exons, this gene works in growth, the most neural
Growing, some sudden changes on this gene are it has been reported that congenital cataract can be caused.Although it have been found that more than 40 base
Because upper a lot of sudden changes can cause congenital cataract, but still there are many diseases because of the brightest, congenital cataract is ground
Study carefully and still need deeply.
Summary of the invention
The present invention is to detect, based on inventor, the new shearing site finding to identify congenital cataract Disease-causing gene
Suddenly change and make.
According to a first aspect of the present invention, the present invention provides a kind of saltant type EPHA2 gene, and it has a SEQ ID NO:
Sequence shown in 1.According to one embodiment of present invention, the sequence shown in described SEQ ID NO:1 is to separate
, c.2825+1G it can produce by making wild type EPHA2 gene > A suddenlys change formation.Detect this mutated genes,
Congenital cataract biological specimen can be gone out with examination, can be used for early screening congenital cataract pathogenic mutation carrier, enter
And carrier fall ill before carry out early intervention treatment it can also be used to the molecular diagnosis of patients with congenital cataract and with phase
The Differential Diagnosis of related disorders, it is also possible to for manufacturing of congenital cataract medicine.Detect this mutated genes
With detection examination congenital cataract, there is quick, accurate, efficient, easy, early diagnostic rate advantages of higher, detection knot
Fruit can be that the exploitation preparation of the early diagnosis of congenital cataract, Differential Diagnosis and medicine provides scientific basis.
According to a second aspect of the present invention, the present invention provide said gene congenital cataractous early screening, diagnosis and
/ or prepare the purposes in treatment congenital cataract medicine.Detect this mutated genes, congenital cataract can be gone out with examination
Biological specimen, can be used for early screening congenital cataract pathogenic mutation carrier, and then carried out before carrier is fallen ill
Early intervention is treated it can also be used to the molecular diagnosis of patients with congenital cataract and the Differential Diagnosis with relevant disease, also may be used
For manufacturing of congenital cataract medicine.Detect this mutated genes to detect examination congenital cataract
Having quick, accurate, efficient, easy, early diagnostic rate advantages of higher, testing result can be congenital cataract
The exploitation preparation of early diagnosis, Differential Diagnosis and medicine provides scientific basis.
According to a third aspect of the present invention, the present invention provides the polypeptide of any of the above-described gene code.According to the present invention one
Embodiment, this polypeptide has the aminoacid sequence shown in SEQ ID NO:2.Optional, described SEQ ID NO:2
Shown aminoacid sequence occurs p.D942E* to be formed due to wild type EPHA2 albumen.Detect this mutated genes
Polypeptide/the albumen of coding, can go out congenital cataract biological specimen with examination, can be used for early screening congenital cataract and causes
Sick carriers of mutation, and then before carrier is fallen ill, carry out early intervention treatment it can also be used to patients with congenital cataract
Molecular diagnosis and with the Differential Diagnosis of relevant disease, it is also possible to the preparation for the medicine of congenital cataract is opened
Send out.The albumen detecting this mutated genes coding has quick, accurate, efficient, simple with detection examination congenital cataract
Just, early diagnostic rate advantages of higher, testing result can be the early diagnosis of congenital cataract, Differential Diagnosis and treatment
The exploitation preparation of medicine provides scientific basis.
According to a fourth aspect of the present invention, the present invention provides any of the above-described polypeptide at congenital cataractous early screening, examines
The purposes in congenital cataract medicine is treated in disconnected and/or preparation.
According to a fifth aspect of the present invention, the present invention provides a kind of method of examination congenital cataract biological specimen, the party
Method comprises the following steps: obtain the nucleic acid of described biological specimen；To the region comprising EPHA2 gene in described nucleic acid
Carry out sequencing, it is thus achieved that target sequence；Described target sequence is compared with any of the above-described saltant type EPHA2 gene
Relatively, both unanimously indicate described biological specimen to be congenital cataract biological specimen.According to one embodiment of present invention,
Described the region comprising EPHA2 gene in nucleic acid is carried out sequencing, it is thus achieved that target sequence, including: utilize and draw
Thing expands described nucleic acid, it is thus achieved that amplified production, the sequence of described primer such as SEQ ID NO:3 and SEQ ID NO:4
Shown in, described amplified production is carried out sequencing, to obtain target sequence.By the present invention this on the one hand or arbitrary
The method of biological this product of the examination congenital cataract in embodiment, can filter out congenital white interior effectively, efficiently
Barrier biological specimen.
According to a sixth aspect of the present invention, the present invention provides the system of a kind of examination congenital cataract biological specimen, and this is
System is in order to implement all or part of step of the invention described above screening method on the one hand or in any embodiment, this system
Including: nucleic acid acquisition device, for obtaining the nucleic acid of described biological specimen；Sequencing device, obtains with described nucleic acid
Device is connected, for the region comprising EPHA2 gene in the nucleic acid in described nucleic acid acquisition device is carried out sequence survey
Fixed, to obtain target sequence；Gene comparision device, is connected with described sequencing device, for by described sequencing
Target sequence in device and the saltant type EPHA2 gene in the invention described above one side compare, in order to indicate two
Biological specimen when person is consistent is congenital cataract biological specimen.According to one embodiment of present invention, described sequence is surveyed
Determining device and comprise amplification unit, described amplification unit is connected with described nucleic acid acquisition device, in order to utilize described in primer amplification
Nucleic acid in nucleic acid acquisition device, it is thus achieved that amplified production, the sequence of described primer such as SEQ ID NO:3 and SEQ ID NO:
Shown in 4.By the system of biological this product of this examination congenital cataract on the one hand or in any embodiment of the present invention,
Congenital cataract biological specimen can be filtered out effectively, efficiently.
According to a seventh aspect of the present invention, the present invention provides the test kit of a kind of examination congenital cataract biological specimen, should
Test kit includes: the reagent of the saltant type EPHA2 gene being adapted to detect in the invention described above on the one hand any embodiment.
Reagent can be probe, chip, primer and/or the genomic DNA of saltant type EPHA2 gene or cDNA at least
A part, according to one embodiment of present invention, this test kit includes: sequence such as SEQ ID NO:3 and SEQ ID NO:
Primer shown in 4；Optional, this test kit also includes sequencing reagent.Utilize the present invention this one side or arbitrary enforcement
Test kit in example, it is possible to effectively, examination efficiently go out congenital biological sample.
The discovery detection of the new mutation of the gene of the present invention make use of full exon group sequencing technologies, full exon group order-checking skill
Art is to utilize DNA sequence probe to be captured by the exon region in full-length genome, then enters for each exon
The technology of row degree of depth order-checking.Scanning compared to Linkage mapping and known, this technology is convenient, quick, efficient,
Especially for the disease that genetic heterogeneity is higher, be conducive to promoting relevant disease diagnosis and the progress for the treatment of.
The Testing and appraisal of the new mutation of the gene of the present invention have employed the full exon group sequencing technologies of a new generation, based on to one
Individual 5 generation Congenital Cataract Pedigrees have carried out full-length genome exon group sequencing analysis, find that a congenital cataract causes
The new heterozygous mutant of ospc gene EPHA2.The new mutation that the present invention identifies enriches the pathogenic mutation of EPHA2 gene
Collection of illustrative plates, further illustrates the Molecular pathogenesis of congenital cataract, and the early stage Disease-causing gene for congenital cataract sieves
Look into and provide scientific basis with therapeutic intervention.
Accompanying drawing explanation
Above-mentioned and/or the additional aspect of the present invention and advantage are from combining the accompanying drawings below description to embodiment and will become
Substantially with easy to understand, wherein:
Fig. 1 is the flow chart of the method for the examination congenital cataract biological specimen in one embodiment of the present of invention.
Fig. 2 is the flow chart of the method for the examination congenital cataract biological specimen in one embodiment of the present of invention.
Fig. 3 is the structural representation of the system of the examination congenital cataract biological specimen in one embodiment of the present of invention.
Fig. 4 is the structural representation of the system of the examination congenital cataract biological specimen in one embodiment of the present of invention.
Fig. 5 is the pedigree chart in one embodiment of the present of invention.
Fig. 6 is the Sanger sequencing result peak figure in one embodiment of the present of invention.
Fig. 7 is the Sanger sequencing result peak figure in one embodiment of the present of invention.
Fig. 8 is the Sanger sequencing result peak figure in one embodiment of the present of invention.
Fig. 9 is the Sanger sequencing result peak figure in one embodiment of the present of invention.
Detailed description of the invention
Embodiments of the invention are described below in detail, and the example of described embodiment is shown in the drawings, wherein, from start to finish
Same or similar label represents same or similar element or has the element of same or like function.Below with reference to
The embodiment that accompanying drawing describes is exemplary, is only used for explaining the present invention, and is not considered as limiting the invention.Need
To be illustrated, term used herein " first ", " second " or " Part I " etc. describes only for convenience,
It is not intended that instruction or hint relative importance, between being interpreted as, can not there is sequencing relation.Retouching in the present invention
In stating, except as otherwise noted, " multiple " are meant that two or more.In this article, unless otherwise clear and definite
Regulation and limiting, term " is connected ", the term such as " connection " should be interpreted broadly, for example, it may be fix connection, also
Can be to removably connect, or be integrally connected；Can be to be mechanically connected, it is also possible to be electrical connection；It can be direct phase
Even, it is also possible to be indirectly connected to by intermediary, can be the connection of two element internals.
According to one embodiment of present invention, the present invention provides a kind of saltant type EPHA2 gene, and it has a SEQ ID NO:
CDNA sequence shown in 1.The cDNA of wild type EPHA2 gene and protein sequence can obtain from following network address:http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi？REQUEST=GV&DATA=338422&BUI LDS=ALLBUILDS, the saltant type cDNA sequence i.e. SEQ ID NO:1 of EPHA2 gene shows as follows
ATGGAGCTCCAGGCAGCCCGCGCCTGCTTCGCCCTGCTGTGGGGCTGTGCGCTGGCCGCGGCCGCGGCGG
CGCAGGGCAAGGAAGTGGTACTGCTGGACTTTGCTGCAGCTGGAGGGGAGCTCGGCTGGCTCACACACCC
GTATGGCAAAGGGTGGGACCTGATGCAGAACATCATGAATGACATGCCGATCTACATGTACTCCGTGTGC
AACGTGATGTCTGGCGACCAGGACAACTGGCTCCGCACCAACTGGGTGTACCGAGGAGAGGCTGAGCGTA
TCTTCATTGAGCTCAAGTTTACTGTACGTGACTGCAACAGCTTCCCTGGTGGCGCCAGCTCCTGCAAGGA
GACTTTCAACCTCTACTATGCCGAGTCGGACCTGGACTACGGCACCAACTTCCAGAAGCGCCTGTTCACC
AAGATTGACACCATTGCGCCCGATGAGATCACCGTCAGCAGCGACTTCGAGGCACGCCACGTGAAGCTGA
ACGTGGAGGAGCGCTCCGTGGGGCCGCTCACCCGCAAAGGCTTCTACCTGGCCTTCCAGGATATCGGTGC
CTGTGTGGCGCTGCTCTCCGTCCGTGTCTACTACAAGAAGTGCCCCGAGCTGCTGCAGGGCCTGGCCCAC
TTCCCTGAGACCATCGCCGGCTCTGATGCACCTTCCCTGGCCACTGTGGCCGGCACCTGTGTGGACCATG
CCGTGGTGCCACCGGGGGGTGAAGAGCCCCGTATGCACTGTGCAGTGGATGGCGAGTGGCTGGTGCCCAT
TGGGCAGTGCCTGTGCCAGGCAGGCTACGAGAAGGTGGAGGATGCCTGCCAGGCCTGCTCGCCTGGATTT
TTTAAGTTTGAGGCATCTGAGAGCCCCTGCTTGGAGTGCCCTGAGCACACGCTGCCATCCCCTGAGGGTG
CCACCTCCTGCGAGTGTGAGGAAGGCTTCTTCCGGGCACCTCAGGACCCAGCGTCGATGCCTTGCACACG
ACCCCCCTCCGCCCCACACTACCTCACAGCCGTGGGCATGGGTGCCAAGGTGGAGCTGCGCTGGACGCCC
CCTCAGGACAGCGGGGGCCGCGAGGACATTGTCTACAGCGTCACCTGCGAACAGTGCTGGCCCGAGTCTG
GGGAATGCGGGCCGTGTGAGGCCAGTGTGCGCTACTCGGAGCCTCCTCACGGACTGACCCGCACCAGTGT
GACAGTGAGCGACCTGGAGCCCCACATGAACTACACCTTCACCGTGGAGGCCCGCAATGGCGTCTCAGGC
CTGGTAACCAGCCGCAGCTTCCGTACTGCCAGTGTCAGCATCAACCAGACAGAGCCCCCCAAGGTGAGGC
TGGAGGGCCGCAGCACCACCTCGCTTAGCGTCTCCTGGAGCATCCCCCCGCCGCAGCAGAGCCGAGTGTG
GAAGTACGAGGTCACTTACCGCAAGAAGGGAGACTCCAACAGCTACAATGTGCGCCGCACCGAGGGTTTC
TCCGTGACCCTGGACGACCTGGCCCCAGACACCACCTACCTGGTCCAGGTGCAGGCACTGACGCAGGAGG
GCCAGGGGGCCGGCAGCAAGGTGCACGAATTCCAGACGCTGTCCCCGGAGGGATCTGGCAACTTGGCGGT
GATTGGCGGCGTGGCTGTCGGTGTGGTCCTGCTTCTGGTGCTGGCAGGAGTTGGCTTCTTTATCCACCGC
AGGAGGAAGAACCAGCGTGCCCGCCAGTCCCCGGAGGACGTTTACTTCTCCAAGTCAGAACAACTGAAGC
CCCTGAAGACATACGTGGACCCCCACACATATGAGGACCCCAACCAGGCTGTGTTGAAGTTCACTACCGA
GATCCATCCATCCTGTGTCACTCGGCAGAAGGTGATCGGAGCAGGAGAGTTTGGGGAGGTGTACAAGGGC
ATGCTGAAGACATCCTCGGGGAAGAAGGAGGTGCCGGTGGCCATCAAGACGCTGAAAGCCGGCTACACAG
AGAAGCAGCGAGTGGACTTCCTCGGCGAGGCCGGCATCATGGGCCAGTTCAGCCACCACAACATCATCCG
CCTAGAGGGCGTCATCTCCAAATACAAGCCCATGATGATCATCACTGAGTACATGGAGAATGGGGCCCTG
GACAAGTTCCTTCGGGAGAAGGATGGCGAGTTCAGCGTGCTGCAGCTGGTGGGCATGCTGCGGGGCATCG
CAGCTGGCATGAAGTACCTGGCCAACATGAACTATGTGCACCGTGACCTGGCTGCCCGCAACATCCTCGT
CAACAGCAACCTGGTCTGCAAGGTGTCTGACTTTGGCCTGTCCCGCGTGCTGGAGGACGACCCCGAGGCC
ACCTACACCACCAGTGGCGGCAAGATCCCCATCCGCTGGACCGCCCCGGAGGCCATTTCCTACCGGAAGT
TCACCTCTGCCAGCGACGTGTGGAGCTTTGGCATTGTCATGTGGGAGGTGATGACCTATGGCGAGCGGCC
CTACTGGGAGTTGTCCAACCACGAGGTGATGAAAGCCATCAATGATGGCTTCCGGCTCCCCACACCCATG
GACTGCCCCTCCGCCATCTACCAGCTCATGATGCAGTGCTGGCAGCAGGAGCGTGCCCGCCGCCCCAAGT
TCGCTGACATCGTCAGCATCCTGGACAAGCTCATTCGTGCCCCTGACTCCCTCAAGACCCTGGCTGACTT
TGACCCCCGCGTGTCTATCCGGCTCCCCAGCACGAGCGGCTCGGAGGGGGTGCCCTTCCGCACGGTGTCC
GAGTGGCTGGAGTCCATCAAGATGCAGCAGTATACGGAGCACTTCATGGCGGCCGGCTACACTGCCATCG
AGAAGGTGGTGCAGATGACCAACGAATAA。
According to one embodiment of present invention, the cDNA sequence shown in described SEQ ID NO:1 is due to wild type
C.2825+1G EPHA2 gene occurs > A sudden change formation.C.2825+1G > A represents wild type EPHA2 gene cDNA
After 2825th bit base of sequence on 1 intron (intron sequences is not present in above-mentioned cDNA sequence)
G has been mutated into A, causes variable sheer, and the cDNA sequence after causing there occurs change, i.e. shows above-mentioned cDNA
106 bases after in sequence are substituted by four bases of ATAA (in SEQ ID NO:1, overstriking shows).
Inventor by detection analyze find, when occurring c.2825+1G in EPHA2 genic mutation type > A and/or
During p.D942E* sudden change, congenital cataract in hgher efficiency of detection biological specimen.By detection, there is this sudden change
Whether EPHA2 genic mutation type exists in biological specimen, can predict that belonging to biological specimen, individuality is accurately and effectively
No suffer from congenital cataract.It should be noted that be not yet related to the EPHA2 gene of present invention proposition at present
C.2825+1G > A sudden change causes the report of congenital cataract.
Detect this mutated genes, congenital cataract biological specimen can be gone out with examination, can be used for early screening congenital in vain
Cataract pathogenic mutation carrier, so before carrier is fallen ill, carry out early intervention treatment it can also be used to congenital white in
The molecular diagnosis of barrier patient and with the Differential Diagnosis of relevant disease, it is also possible to for the opening of medicine of congenital cataract
Send out.Detect this mutated genes with detection examination congenital cataract have quick, accurate, efficient, easy, examine in early days
Disconnected rate advantages of higher, testing result can be the exploitation of the early diagnosis of congenital cataract, Differential Diagnosis and medicine
Scientific basis is provided.
According to some embodiments of the present invention, the present invention provides said mutation type gene to sieve in congenital cataractous early stage
Look into, diagnose and/or prepare the purposes in congenital cataract medicine.
According to one embodiment of present invention, the mutated genes coding during the present invention provides any of the above-described embodiment is many
Peptide.
According to one embodiment of present invention, this polypeptide has the aminoacid sequence shown in SEQ ID NO:2.Saltant type
The aminoacid sequence of EPHA2 gene i.e. SEQ ID NO:2 shows as follows
MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVC
NVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFT
KIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCPELLQGLAH
FPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGF
FKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTP
PQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSG
LVTSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGF
SVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIGGVAVGVVLLLVLAGVGFFIHR
RRKNQRARQSPEDVYFSKSEQLKPLKTYVDPHTYEDPNQAVLKFTTEIHPSCVTRQKVIGAGEFGEVYKG
MLKTSSGKKEVPVAIKTLKAGYTEKQRVDFLGEAGIMGQFSHHNIIRLEGVISKYKPMMIITEYMENGAL
DKFLREKDGEFSVLQLVGMLRGIAAGMKYLANMNYVHRDLAARNILVNSNLVCKVSDFGLSRVLEDDPEA
TYTTSGGKIPIRWTAPEAISYRKFTSASDVWSFGIVMWEVMTYGERPYWELSNHEVMKAINDGFRLPTPM
DCPSAIYQLMMQCWQQERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSGSEGVPFRTVS
EWLESIKMQQYTEHFMAAGYTAIEKVVQMTNE。
According to one embodiment of present invention, the aminoacid sequence shown in described SEQ ID NO:2 is due to wild type
EPHA2 gene occurs p.D942E* to be formed.P.D942E* represents the protein sequence albumen of coding (wild type) the
The aspartic acid of 942 is substituted by glutamic acid (SEQ ID NO:2 is shown in bold), simultaneously the most original amino
Acid terminates translation in advance due to the appearance of new termination codon, causes the truncate of this protein.
Inventor by detection analyze find, when occurring c.2825+1G in EPHA2 genic mutation type > A and/or
During p.D942E* sudden change, congenital cataract in hgher efficiency of detection biological specimen.By detection, there is this sudden change
Whether EPHA2 genic mutation type exists in biological specimen, can predict that belonging to biological specimen, individuality is accurately and effectively
No suffer from congenital cataract.It should be noted that be not yet related to the EPHA2 gene of present invention proposition at present
C.2825+1G > A and/or p.D942E* sudden change causes the report of congenital cataract.
Detect the polypeptide/albumen of this mutated genes coding, congenital cataract biological specimen can be gone out with examination, can be used for early
Phase examination congenital cataract pathogenic mutation carrier, and then carried out early intervention treatment before carrier is fallen ill, it is possible to
For patients with congenital cataract molecular diagnosis and with the Differential Diagnosis of relevant disease, it is also possible to for congenital cataract
The manufacturing of medicine.The albumen detecting this mutated genes coding has with detection examination congenital cataract soon
Speed, accurate, efficient, easy, early diagnostic rate advantages of higher, testing result can be to examine the early stage of congenital cataract
Disconnected, Differential Diagnosis and medicine offer scientific basis is provided.
According to some embodiments of the present invention, the present invention provides the polypeptide in any of the above-described embodiment congenital cataractous
Purposes in early screening, diagnosis and/or medicine exploitation.
According to one embodiment of present invention, as it is shown in figure 1, the present invention provides a kind of examination congenital cataract biology sample
This method, the method comprises the following steps: S10 obtains the nucleic acid of described biological specimen；S20 is in described nucleic acid
The region comprising EPHA2 gene carries out sequencing, it is thus achieved that target sequence；S30 by described target sequence with above-mentioned
One saltant type EPHA2 gene compares, and both unanimously indicate described biological specimen to be congenital cataract biological specimen.
Unanimously refer to both alleged that sequence is consistent, refers in particular to mutant nucleotide sequence consistent, such as, compared with wild type cDNA, the two
CDNA sequence the 2825th bit base after 106 bases be ATAA.
According to one embodiment of present invention, as in figure 2 it is shown, S20 includes: S201 utilizes nucleic acid described in primer amplification,
Obtaining amplified production, the sequence of described primer is as shown in SEQ ID NO:3 and SEQ ID NO:4, and S203 is to described
Amplified production carries out sequencing, to obtain target sequence.Congenital in vain by the examination in any embodiment of the present invention
The method of biological this product of cataract, can filter out congenital cataract biological specimen effectively, efficiently.
According to one embodiment of present invention, as it is shown on figure 3, the present invention provides a kind of examination congenital cataract biology sample
This system 1000, this system is in order to implement the whole of the invention described above screening method on the one hand or in any embodiment
Or part steps, this system includes: nucleic acid acquisition device 100, for obtaining the nucleic acid of described biological specimen；Sequence is surveyed
Determine device 200, be connected with described nucleic acid acquisition device 100, in the nucleic acid in described nucleic acid acquisition device 100
The region comprising EPHA2 gene carry out sequencing, to obtain target sequence；Gene comparision device 300, with institute
State sequencing device 200 to be connected, for by the target sequence in described sequencing device 200 and the invention described above one
Saltant type EPHA2 gene in aspect compares, and biological specimen during in order to indicate both consistent is congenital white interior
Barrier biological specimen.Unanimously refer to both alleged that sequence is consistent, refers in particular to mutant nucleotide sequence consistent, such as, with wild type cDNA
Comparing, 106 bases after the 2825th bit base of the cDNA sequence of the two are ATAA.
According to one embodiment of present invention, as shown in Figure 4, described sequencing device 200 includes: amplification unit 210,
Described amplification unit 210 is connected with described nucleic acid acquisition device 100, in order to utilize nucleic acid acquisition device described in primer amplification
Nucleic acid in 100, it is thus achieved that amplified production, the sequence of described primer such as SEQ ID NO:3 and SEQ ID NO:4 institute
Show；And order-checking unit 220, described order-checking unit 220 is connected with described amplification unit 210, in order to described amplification
Amplified production in unit 210 carries out sequencing, to obtain target sequence.By the sieve in any embodiment of the present invention
Look into the system of biological this product of congenital cataract, congenital cataract biological specimen can be filtered out effectively, efficiently.
According to one embodiment of present invention, the present invention provides the test kit of a kind of examination congenital cataract biological specimen,
This test kit includes: the reagent of the saltant type EPHA2 gene being adapted to detect in the invention described above on the one hand any embodiment.
Reagent can be probe, chip, primer and/or the genomic DNA of saltant type EPHA2 gene or cDNA at least
A part, according to one embodiment of present invention, this test kit includes: such as SEQ ID NO:3 and SEQ ID NO:
Sequence shown in 4；Optional, this test kit also includes sequencing reagent.Utilize the test kit in any embodiment of the present invention,
Can effectively, examination efficiently go out congenital biological sample.
The discovery detection of the new mutation of the gene of the present invention make use of full exon group sequencing technologies, full exon group order-checking skill
Art is to utilize DNA sequence probe to be captured by the exon region in full-length genome, then enters for each exon
The technology of row degree of depth order-checking.Scanning compared to Linkage mapping and known, this technology is convenient, quick, efficient,
Especially for the disease that genetic heterogeneity is higher, be conducive to promoting relevant disease diagnosis and the progress for the treatment of.
The Testing and appraisal of the new mutation of the gene of the present invention have employed the full exon group sequencing technologies of a new generation, based on to one
Individual 5 generation Congenital Cataract Pedigrees have carried out full-length genome exon group sequencing analysis, find that a congenital cataract causes
The new heterozygous mutant of ospc gene EPHA2.The new mutation that the present invention identifies enriches the pathogenic mutation of EPHA2 gene
Collection of illustrative plates, further illustrates the Molecular pathogenesis of congenital cataract, and the early stage Disease-causing gene for congenital cataract sieves
Look into and provide scientific basis with therapeutic intervention.
Below with reference to specific embodiment, the present invention will be described, it should be noted that these embodiments are only explanation
Property, and be not considered as limiting the invention.If not specializing, the technological means employed in embodiment is this
Conventional means known to skilled person, is referred to " Molecular Cloning: A Laboratory guide " third edition or Related product
Carrying out, the reagent used and product are also and can be obtained by conventional commercial.The various processes not described in detail and method
It is conventional method as known in the art, the source of agents useful for same, trade name and be necessary to list its constituent person,
All indicating when occurring first, identical reagent used is if no special instructions, all identical with the content indicated first thereafter.
Embodiment one
1. sample collection
Inventor have collected the Congenital Cataract Pedigree in only one China Anhui, and the patient in family shows congenital white interior
The phenotype of barrier, in autosomal dominant inheritance, AD pattern, pedigree chart is as it is shown in figure 5, expression patient solid in figure, hollow
Expression normal person, II1, III11, IV2 and IV8 are the samples having surveyed exon.We acquire this family part
The peripheral blood of member, and extract DNA, choose 4 members therein (3 patients, 1 normal person) and carry out exon
Order-checking, the participant of be provided with blood sample all signs genus Informed Consent Form, and obtains Ethics Committee's approval.
2. chip design, library construction and high-flux sequence
Inventor with NimbleGen SeqCap EZ Human Exome Library v2.0 (NimbleGen, Madison,
WI, USA) combine Solexa high throughput sequencing technologies the exon group sequence of 4 members in this family is surveyed
Sequence, specific as follows:
1) utilize capture chip (NimbleGen SeqCap EZ Exome (44M)) to the exon of genome, montage
Site and neighbouring intron sequences capture.
2) genomic DNA is broken at random the fragment of about 200-300bp, subsequently on fragment two ends connect respectively
Joint, preparation library (the Illumina/Solexa standard that seeing http://www.illumina.com/ provides builds storehouse description).
3) through linear amplification and the custom capture of ligation-mediated PCR (LM-PCR) after library is purified
Array carries out hybridization enrichment, then carries out upper machine order-checking after the linear amplification of LM-PCR.Order-checking platform is Illumina
Hiseq 2000, reads a length of 100bp.
3. variation detects, annotates and compare with data base
1) initial data obtained after order-checking is processed by Illumina basecalling Software 1.7.Then filter
Low quality reads pollutes reads with comprising joint.Use Burrows Wheeler Aligner (BWA) [H.Li and R.
Durbin,Fast and accurate short read alignment with Burrows-Wheeler transform,
Bioinformatics., 25 (2009) 1754-1760.] and Short Oligonucleotide Analysis Package
(SOAPaligner 2.21)【R.Li,C.Yu,Y.Li,T.W.Lam,S.M.Yiu,K.Kristiansen,and J.Wang.
SOAP2:an improved ultrafast tool for short read alignment,Bioinformatics.,25(2009)
1966-1967.] by high-quality reads comparison to reference to genome (hg19), it is thus achieved that in comparison to genome uniquely than
To reads.Then result SOAPsnp1.05[R.Li to SOAP comparison, Y.Li, X.Fang, H.Yang, J.Wang,
K.Kristiansen,and J.Wang,SNP detection for massively parallel whole-genome
Resequencing, Genome Res., 19 (2009) 1124-1132.] carry out the detection of SNP, the knot to BWA comparison
Fruit Genome Analysis Tool Kit (GATK1.4) [M.A.DePristo, E.Banks, R.Poplin, K.V.
Garimella,J.R.Maguire,C.Hartl,A.A.Philippakis,A.G.del,M.A.Rivas,M.Hanna,A.
McKenna,T.J.Fennell,A.M.Kernytsky,A.Y.Sivachenko,K.Cibulskis,S.B.Gabriel,D.
Altshuler,and M.J.Daly,A framework for variation discovery and genotyping using
Next-generation DNA sequencing data, Nat.Genet., 43 (2011) 491-498.] carry out the detection of indel.
2) for SNP and the indel variation of detection, we annotate, i.e. genomic context residing for definitive variation,
And the type of variation.We pay close attention to nonsynonymous mutation, and acceptor splicing site/donor site sudden change and coding region insert and lack prominent
Become the sudden change that this three class is the most relevant to disease.By variation and dbSNP data base, the thousand human genome numbers of detection
Compare according to storehouse and ESP database public database, remain the variation (MAF≤0.01) of low frequency.According to
Hereditary pattern (autosomal dominant inheritance, AD), have chosen in patient the wildest normal site isozygotied for sudden change heterozygosis,
Based on above-mentioned filtration, we have carried out the scanning of known to remaining sudden change, find and only find EPHA2 gene
On have the sudden change of shearing site, we have carried out Sanger checking, the Sanger of the sample of 4 members to this sudden change
The result is as shown in Figure 6.
3) being analyzed by above, we are found that the heterozygous mutant do not reported on the EPHA2 gene of patient
C.2825+1G > A, p.D942E*, this sudden change is sudden change heterozygosis in patients, is wild isozygotying in normal person, we
Follow-up this site of other members in family is carried out sanger checking, the Sanger checking of other members of this family
Result as Figure 7-9, result demonstrate this sudden change be divided into disease in family from.This sudden change simultaneously is in our prior
Do not exist with in 1000 exon data bases of same exon trapping probe order-checking.
Sudden change explanation:
C.2825+1G > after A:cDNA the 2825th bit base 1 intron (intron sequences is not present in cDNA
In sequence) on G be mutated into A, cause variable sheer, the cDNA sequence after causing there occurs the biggest change
Change (106 bases after in this instance, causing are substituted by tetra-bases of ATAA).
The aspartic acid that p.D942E*: protein sequence is the 942nd is substituted by glutamic acid, simultaneously the most original aminoacid
Terminate translation due to the appearance of new termination codon in advance, cause the truncate of protein.
Embodiment two
1. prepared by sample
Gather member's peripheral blood in family, utilize the genomic DNA in conventional phenol-chloroform method extracting peripheral blood leucocyte,
Utilize concentration and the purity of spectrophotometer measurement DNA, the OD260/OD280 of each specimen genomic DNA of gained
Being respectively positioned between 1.7-2.0, concentration is no less than 200ng/ μ l, and total amount is no less than 30 μ g.
2. disease mutation site detection
Respectively to having surveyed 3 clinical samples of exon and 1 normal sample in family and other are ready to donate sample
Family member carries out Sanger order-checking, and including design of primers, PCR expands, product purification, and order-checking obtains sequence, root
Saltant type or wild type, the dependency between checking sudden change and congenital cataract is belonged to according to sequencing results.Specifically
Method step is as follows:
1) DNA extraction:
Respectively the peripheric venous blood participating in research worker is extracted genomic DNA, light splitting light according to the method for embodiment one
Degree measurement DNA content.
2) design of primers and PCR reaction
Design of primers reference man genoid data unit sequence storehouse hg19/build36.3, is specifically shown in down.
A) primer sequence:
Forward primer: 5'-CGGCACATAGCCCTCAGTAA-3'(SEQ ID NO:3)
Reverse primer: 5'-GAGGGGCAGCAGTAGTTACA-3'(SEQ ID NO:4)
B) reaction system: 25 μ L
C) reaction condition:
3) DNA sequencing is carried out with the 3730 of ABI after purification by step 2 obtains pcr amplification product.
In this family, patient is the sudden change heterozygote of pathogenic mutation, and normal person is wild homozygote.Therefore we
Thinking on EPHA2 gene c.2825+1G > A sports a pathogenic mutation of congenital cataract.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " concrete example ",
Or specific features, material or the feature that the description of " some examples " etc. means to combine this embodiment or example describes is contained in
In at least one embodiment of the present invention or example.In this manual, the schematic representation of above-mentioned term is not necessarily referred to
Be identical embodiment or example.And, the specific features of description, structure, material or feature can be any
One or more embodiments or example combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: do not taking off
In the case of the principles and objective of the present invention, these embodiments can be carried out multiple change, revise, replace and modification,
The scope of the present invention is limited by claim and equivalent thereof.
Claims (10)
1. a saltant type EPHA2 gene, it is characterised in that there is the sequence shown in SEQ ID NO:1；
Optional, the sequence shown in described SEQ ID NO:1 is that c.2825+1G wild type EPHA2 gene occurs > A dashes forward
It is deformed into.
2. the gene of claim 1 congenital cataractous early screening, diagnosis and/or preparation treatment congenital white in
Purposes in barrier medicine.
3. the polypeptide of the gene code of claim 1.
4. the polypeptide of claim 3, it is characterised in that there is the aminoacid sequence shown in SEQ ID NO:2；
Optional, the aminoacid sequence shown in described SEQ ID NO:2 is that wild type EPHA2 albumen occurs p.D942E*
Sudden change is formed.
5. the polypeptide of claim 3 or 4 in congenital cataractous early screening, diagnosis and/or is treated congenital in preparation
Purposes in cataract medicine.
6. the method for an examination congenital cataract biological specimen, it is characterised in that comprise the following steps:
Obtain the nucleic acid of described biological specimen；
The region comprising EPHA2 gene in described nucleic acid is carried out sequencing, it is thus achieved that target sequence, optional, including:
Utilize nucleic acid described in primer amplification, it is thus achieved that amplified production, the sequence of described primer such as SEQ ID NO:3 and SEQ
Shown in ID NO:4,
Described amplified production is carried out sequencing, to obtain target sequence；
Being compared by the gene of described target sequence with claim 1, both unanimously indicate described biological specimen to be congenital
Cataract biological specimen.
7. the system of an examination congenital cataract biological specimen, it is characterised in that including:
Nucleic acid acquisition device, for obtaining the nucleic acid of described biological specimen；
Sequencing device, is connected with described nucleic acid acquisition device, for the bag in the nucleic acid in described nucleic acid acquisition device
Region containing EPHA2 gene carries out sequencing, to obtain target sequence；
Gene comparision device, is connected with described sequencing device, for by the target sequence in described sequencing device with
The gene of claim 1 compares, and biological specimen during in order to indicate both consistent is congenital cataract biological specimen.
8. the system of claim 7, it is characterised in that described sequencing device comprises amplification unit,
Described amplification unit is connected with described nucleic acid acquisition device, in order to utilize the core in nucleic acid acquisition device described in primer amplification
Acid, it is thus achieved that amplified production, the sequence of described primer is as shown in SEQ ID NO:3 and SEQ ID NO:4.
9. the test kit of an examination congenital cataract biological specimen, it is characterised in that including:
It is adapted to detect for the reagent of the gene of claim 1.
10. the test kit of claim 9, it is characterised in that including:
Sequence as shown in SEQ ID NO:3 and SEQ ID NO:4.
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Non-Patent Citations (2)
| Title |
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| JUAN BU,ET AL: "A novel splice donor site mutation in EPHA2 caused congenital cataract in a Chinese family", 《INDIAN JOURNAL OF OPHTHALMOLOGY》 * |
| 华芮: "先天性白内障的分子遗传学研究", 《中国博士学位论文全文数据库》 * |
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