CN105985963A - CRX gene mutant and application thereof - Google Patents

Abstract

The invention discloses a CRX gene mutant and application thereof, and particularly relates to separated nucleic acid of an encoded CRX gene mutant, separated polypeptide and a method, system and kit for screening biological samples likely to suffer from cone and rod cell malnutrition. Compared with SEQ ID NO:1, according to the separated nucleic acid of the encoded CRX gene mutant, c.766C is larger than T mutation. Whether the novel mutant exists in the biological samples or not is detected, and therefore whether the biological samples are likely to suffer from cone and rod cell malnutrition or not can be effectively detected.

Description

CRX gene mutation body and application thereof

Technical field

The present invention relates to CRX gene mutation body and application thereof.In particular it relates to separate the core of coding CRX mutant Acid, separate polypeptide, screening be susceptible to suffer from cone rod cell muscular dystrophy biological sample system, for screening be susceptible to suffer from the cone The test kit of the biological sample of rod cell muscular dystrophy, construct, reconstitution cell and the method building medicaments sifting model.

Background technology

Cone rod cell malnutrition (cone-rod dystrophies, CORDs) is one group of genetic gradual retina Disease, sickness rate is 1/40000.I.e. there is dysfunction, subsequently in early days in morbidity in the cone cell that is mainly characterized by of CORDs There is functional deterioration in rod cell.The clinical manifestation of such disease includes photophobia, visual deterioration, color defect and central scotoma. There is nystagmus symptom in some patient.Cone cell afunction or to be badly damaged be the typical case of CORDs on electroretinogram Mark.Generally, the most just observe that rod cell is impaired after cone cell function generation obstacle.Under extreme case, these are progressive Property symptom with widely, serious retinal pigment calm, and central authorities and peripheral retina atrophy occur.To later stage, base May be difficult to distinguish such disease and retinitis pigmentosa in clinical symptoms.

Genetic heterogeneity and the phenotype heterogeneity of CORDs are the highest.Such disease not only difficult diagnosis because its symptom is overlapping, And the different sudden changes of same gene also result in phenotype difference.CORDs gets final product autosomal dominant inheritance, AD (adCORD), Also can autosomal recessive (arCORD) or X-linkage inheritance (xlCORD).Up to now, the sudden change of at least 26 genes It is in the news relevant to the CORD of multi-form, but still has many diseases because of the brightest.Thus, at present to cone rod cell The research of muscular dystrophy especially its Disease-causing gene still needs deeply.

Summary of the invention

It is contemplated that at least solve one of technical problem present in prior art.To this end, it is an object of the present invention to carry Go out a kind of to be susceptible to suffer from the means of biological sample of cone rod cell muscular dystrophy by Effective selection.

The present invention is that following work based on inventor completes:

Inventor finds, in the research that current Disease-causing gene and pathogenic mutation excavate, and the full exon group sequencing of many uses.Entirely Exon group order-checking be to utilize special DNA sequence probe to be captured by the exon region in full-length genome, then for Each exon carries out the technology of degree of depth order-checking.The order-checking of exon group is utilized to find out the case of mendelian inheritance disease Disease-causing gene very Many, such as 2009 U.S. Sarah B Ng et al. utilize exon group to check order successfully to orient the gene of Miller syndrome DHODH(Ng SB,et al.Exome sequencing identifies the cause of a mendelian disorder.Nat Genet, 2009,42(1):30–35).China Wang in 2010 et al. utilizes the order-checking of exon group to be found that the new of cerebellar ataxia Mutant gene TGM6 (Wang JL, et al.TGM6identified as a novel causative gene of spinocerebellar ataxias using exome sequencing.Brain,2010,133(Pt 12):3510–3518.).Along with exon group sequencing technologies Extensively application and day by day ripe, a collection of new Disease-causing gene (sudden change) is found in succession, has greatly promoted relevant disease to examine Progress that is disconnected and that treat.

This is because, human exonic organizes sequence and only accounts for the 1% of the whole genome sequence of the mankind, about 30Mb, including 180,000 The exon of individual left and right, it is estimated that human disease's sudden change of 85% is all located on this protein coding sequence of 1%.Therefore, The exon group of various Diseases is carried out sequencing analysis, and targeted is maximally related with disease " exome " region, catches Catch is most of pathogenic mutation information of disease.Exon sequencing technologies, as a kind of impartial detection method, becomes the most Merit it is applied in the detection of various pathogenic mutation.

Thus, inventor, for the ill family of adCORD occuping southern china collected, is checked order by exon group The method of associating Sanger sequence verification carries out pathogenic mutation excavation and checking, finally determines cone rod cell malnutrition One new pathogenic mutation CRX gene of disease is c.766C > T sudden change.

And then, according to the first aspect of the invention, the present invention proposes the nucleic acid of the coding CRX mutant of a kind of separation.Root According to embodiments of the invention, c.766C described nucleic acid, compared with SEQ ID NO:1, have > T sudden change, i.e. relative to wild Type CRX gene, cDNA sequence the 766th bit base C of the CRX gene mutation body of the present invention sports T.Root According to embodiments of the invention, inventor determines the mutant of CRX gene, this mutant and cone rod cell malnutrition The morbidity of disease is closely related, thus by detecting whether this mutant exists in biological sample, can effectively detect biological sample Whether product are susceptible to suffer from cone rod cell muscular dystrophy.

According to the second aspect of the invention, the present invention proposes the polypeptide of a kind of separation.According to embodiments of the invention, with SEQ ID NO:2 compares, the polypeptide of described separation have p.Q256X sudden change, i.e. this sudden change is due to c.766C > T sudden change and draw Rise, specifically, relative to wild type CRX, the aminoacid sequence of the polypeptide (i.e. CRX mutant) of the separation of the present invention 256th amino acids Q-spoiling is X.By whether detection biological sample expresses this polypeptide, can effectively detect biology Whether sample is susceptible to suffer from cone rod cell muscular dystrophy.

According to the third aspect of the invention we, the present invention proposes a kind of screening and is susceptible to suffer from the biological sample of cone rod cell muscular dystrophy The system of product.According to embodiments of the invention, this system includes: nucleic acid-extracting apparatus, and described nucleic acid-extracting apparatus is for from institute State extraction from biological material sample of nucleic acid；Nucleotide sequence determines that device, described nucleotide sequence determine device and described nucleic acid-extracting apparatus It is connected, for described sample of nucleic acid is analyzed, in order to determine the nucleotide sequence of described sample of nucleic acid；Judgment means, described With described nucleotide sequence, judgment means determines that device is connected, in order to nucleotide sequence based on described sample of nucleic acid or its complementary series, Compared with SEQ ID NO:1, if having c.766C > T sudden change, it is judged that it is thin whether described biological sample is susceptible to suffer from cone retinal rod Born of the same parents' muscular dystrophy.Utilize this system, it is possible to screening is susceptible to suffer from the biological sample of cone rod cell muscular dystrophy effectively.

According to the fourth aspect of the invention, the present invention proposes a kind of life being susceptible to suffer from cone rod cell muscular dystrophy for screening The test kit of thing sample.According to embodiments of the invention, this test kit contains: be adapted to detect for the reagent of CRX gene mutation body, Wherein compared with SEQ ID NO:1, c.766C this CRX gene mutation body has > T sudden change.Utilize the reality according to the present invention Execute the test kit of example, it is possible to screening is susceptible to suffer from the biological sample of cone rod cell muscular dystrophy effectively.

According to the fifth aspect of the invention, the invention allows for a kind of construct.According to embodiments of the invention, this construct Comprise the nucleic acid of the coding CRX mutant of foregoing separation.Thus, the construct transformed acceptor cell of the present invention is utilized The reconstitution cell obtained, it is possible to be efficiently used for the medicine of screening treatment cone rod cell muscular dystrophy.

According to the sixth aspect of the invention, the invention allows for a kind of reconstitution cell.According to embodiments of the invention, this restructuring Cell is obtained by foregoing construct transformed acceptor cell.According to some embodiments of the present invention, utilize this Bright reconstitution cell, it is possible to the medicine of screening treatment cone rod cell muscular dystrophy effectively.

According to the seventh aspect of the invention, the invention allows for a kind of method building medicaments sifting model.According to the present invention's Embodiment, the method includes: make at least some of cell of animal express the nucleic acid of foregoing coding CRX mutant. Thus, the medicaments sifting model of the present invention is utilized, it is possible to the medicine of screening treatment cone rod cell muscular dystrophy effectively.

It should be noted that the pathogenic mutation of the adCORD that present invention discover that new Disease-causing gene CRX is c.766C > T, May be used for early screening adCORD pathogenic mutation carrier, and then carry out early intervention treatment before carrier is fallen ill； Can also be used for adCORD molecular diagnosis and with relevant disease Differential Diagnosis, and quickly, accurately, efficiently, easy, Early diagnostic rate is high, and testing result can be the early diagnosis of cone rod cell muscular dystrophy, Differential Diagnosis and the exploitation cone Rod cell muscular dystrophy medicine provides scientific basis.

The additional aspect of the present invention and advantage will part be given in the following description, and part will become bright from the following description Aobvious, or recognized by the practice of the present invention.

Accompanying drawing explanation

Above-mentioned and/or the additional aspect of the present invention and advantage the accompanying drawings below description to embodiment will be apparent from from combining and Easy to understand, wherein:

Fig. 1 show screening according to embodiments of the present invention be susceptible to suffer from the biological sample of cone rod cell muscular dystrophy system and The schematic diagram of its ingredient, wherein,

Fig. 1 I is the system that the screening according to the embodiment of the present invention is susceptible to suffer from the biological sample of cone rod cell muscular dystrophy Schematic diagram,

Fig. 1 II is the schematic diagram of the nucleic acid-extracting apparatus according to the embodiment of the present invention,

Fig. 1 III is the schematic diagram that the nucleotide sequence according to the embodiment of the present invention determines device；

Fig. 2 shows the pedigree chart of cone rod cell muscular dystrophy patient's family according to an embodiment of the invention；

Fig. 3 shows according to one embodiment of the invention, the ophthalmologic examination result of proband in patient's family shown in Fig. 2, wherein,

Fig. 3 A-Fig. 3 B is fundus photography result,

Fig. 3 C-Fig. 3 D is optical coherence tomography result,

Fig. 3 E-Fig. 3 G is fundus autofluorescence result；

Fig. 4 shows according to one embodiment of present invention, strong in the muscular dystrophy patient's family of cone rod cell shown in Fig. 2 Health member and proband, and the CRX gene of the outer normal person of family is c.766C > the representative Sanger order-checking in T mutational site Checking peak figure.

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, the most identical or Similar label represents same or similar element or has the element of same or like function.Describe below with reference to accompanying drawing Embodiment is exemplary, is only used for explaining the present invention, and is not considered as limiting the invention.

CRX gene mutation body

According to the first aspect of the invention, the present invention proposes the nucleic acid of coding CRX mutant of a kind of separation.According to the present invention Embodiment, c.766C described nucleic acid, compared with SEQ ID NO:1, have > T sudden change.

The expression way " nucleic acid of coding CRX mutant " used in this article, refers to and encodes the gene of CRX mutant The type of corresponding nucleic acid substances, i.e. nucleic acid is not particularly limited, and can be any comprising and the encoding gene of CRX mutant Corresponding deoxyribonucleotide and/or the polymer of ribonucleotide, include but not limited to DNA, RNA or cDNA.Root According to a concrete example of the present invention, the nucleic acid of foregoing coding CRX mutant is DNA.Enforcement according to the present invention Example, inventor determines the mutant of CRX gene, and this mutant is closely related with the morbidity of cone rod cell muscular dystrophy, Thus by detecting whether this mutant exists in biological sample, effectively can detect whether biological sample is susceptible to suffer from cone retinal rod The bad disease of cytotrophy, it is also possible to by detecting whether this mutant exists in organism, can predict that organism is effectively No it is susceptible to suffer from cone rod cell muscular dystrophy.

For, in description of the invention and claims, mentioning nucleic acid, it will be appreciated by those skilled in the art that actual including mutually Mend double-strand any one, or two.For convenience, in the present specification and claims, although in most cases only Give a chain, but actually also disclose that another complementary therewith chain.Such as, mention SEQ ID NO:1, actual Including its complementary series.Those skilled in the art are further appreciated that and utilize a chain can detect another chain, and vice versa.

The nucleic acid of this coding CRX mutant, is that present inventor passes through the order-checking of exon group, linkage analysis associating Sanger The new pathogenic mutation of the Disease-causing gene CRX of the cone rod cell muscular dystrophy that the method for sequence verification determines.This causes a disease Mutational site is the most referred.

Wherein, the cDNA of wild type CRX gene has a nucleotide sequence as follows:

ATGATGGCGTATATGAACCCGGGGCCCCACTATTCTGTCAACGCCTTGGCCCTAAGTG GCCCCAGTGTGGATCTGATGCACCAGGCTGTGCCCTACCCAAGCGCCCCCAGGAAGC AGCGGCGGGAGCGCACCACCTTCACCCGGAGCCAACTGGAGGAGCTGGAGGCACTGT TTGCCAAGACCCAGTACCCAGACGTCTATGCCCGTGAGGAGGTGGCTCTGAAGATCA ATCTGCCTGAGTCCAGGGTTCAGGTTTGGTTCAAGAACCGGAGGGCTAAATGCAGGC AGCAGCGACAGCAGCAGAAACAGCAGCAGCAGCCCCCAGGGGGCCAGGCCAAGGCC CGGCCTGCCAAGAGGAAGGCGGGCACGTCCCCAAGACCCTCCACAGATGTGTGTCCA GACCCTCTGGGCATCTCAGATTCCTACAGTCCCCCTCTGCCCGGCCCCTCAGGCTCCCC AACCACGGCAGTGGCCACTGTGTCCATCTGGAGCCCAGCCTCAGAGTCCCCTTTGCCT GAGGCGCAGCGGGCTGGGCTGGTGGCCTCAGGGCCGTCTCTGACCTCCGCCCCCTATG CCATGACCTACGCCCCGGCCTCCGCTTTCTGCTCTTCCCCCTCCGCCTATGGGTCTCCG AGCTCCTATTTCAGCGGCCTAGACCCCTACCTTTCTCCCATGGTGCCCCAGCTAGGGG GCCCGGCTCTTAGCCCCCTCTCTGGCCCCTCCGTGGGACCTTCCCTGGCCCAGTCCCCC ACCTCCCTATCAGGCCAGAGCTATGGCGCCTACAGCCCCGTGGATAGCTTGGAATTCA AGGACCCCACGGGCACCTGGAAATTCACCTACAATCCCATGGACCCTCTGGACTACA AGGATCAGAGTGCCTGGAAGTTTCAGATCTTGTAG (SEQ ID NO:1),

Protein of its coding has an aminoacid sequence as follows:

MMAYMNPGPHYSVNALALSGPSVDLMHQAVPYPSAPRKQRRERTTFTRSQLEELEALFA KTQYPDVYAREEVALKINLPESRVQVWFKNRRAKCRQQRQQQKQQQQPPGGQAKARPA KRKAGTSPRPSTDVCPDPLGISDSYSPPLPGPSGSPTTAVATVSIWSPASESPLPEAQRAGLV ASGPSLTSAPYAMTYAPASAFCSSPSAYGSPSSYFSGLDPYLSPMVPQLGGPALSPLSGPSV GPSLAQSPTSLSGQSYGAYSPVDSLEFKDPTGTWKFTYNPMDPLDYKDQSAWKFQIL(SEQ ID NO:2).

Inventor find CRX gene mutation body compared with SEQ ID NO:1, have c.766C > T sudden change, i.e. relative to Wild type CRX gene, cDNA sequence the 766th bit base C of the CRX gene mutation body of the present invention sports T.Thus, Its coded product, compared with the CRX of wild type, has p.Q256X sudden change, i.e. this sudden change is due to c.766C > T sudden change And cause, specifically, relative to wild type CRX, the aminoacid sequence of the polypeptide (i.e. CRX mutant) of the separation of the present invention Arranging the 256th amino acids Q-spoiling is X.

At present, there is not yet CRX gene c.766C > T sports the phase of the pathogenic mutation of cone rod cell muscular dystrophy Close report.

According to the second aspect of the invention, the present invention proposes the polypeptide of a kind of separation.According to embodiments of the invention, Compared with wild type CRX, the polypeptide of this separation have p.Q256X sudden change, i.e. this sudden change is due to c.766C > T sudden change and draw Rise, specifically, relative to wild type CRX, the aminoacid sequence of the polypeptide (i.e. CRX mutant) of the separation of the present invention the 256 amino acids Q-spoilings are X.According to some concrete examples of the present invention, this polypeptide is by the coding CRX of aforementioned separation The nucleic acid coding of mutant.By whether detection biological sample expresses this polypeptide, can effectively detect biological sample Whether product are susceptible to suffer from cone rod cell muscular dystrophy, it is also possible to by detecting whether these polypeptide exist in organism, Can effectively predict whether organism is susceptible to suffer from cone rod cell muscular dystrophy.

Screening is susceptible to suffer from system and the test kit of the biological sample of cone rod cell muscular dystrophy

According to the third aspect of the invention we, the present invention propose one can effectively implement above-mentioned screening be susceptible to suffer from the cone rod cell battalion Support the system of the method for the biological sample of bad disease.

With reference to Fig. 1, according to embodiments of the invention, what this screening was susceptible to suffer from the biological sample of cone rod cell muscular dystrophy is System 1000 includes that nucleic acid-extracting apparatus 100, nucleotide sequence determine device 200 and judgment means 300.

According to embodiments of the invention, nucleic acid-extracting apparatus 100 is for from extraction from biological material sample of nucleic acid.According to the present invention's Embodiment, the type of biological sample is not particularly restricted, as long as can extract reflection biological sample from this biological sample Whether CRX exists the sample of nucleic acid of sudden change.According to embodiments of the invention, biological sample can be selected from blood of human body, Skin, hypodermic at least one, preferably peripheral blood.Thus, it is possible to be sampled easily and detect such that it is able to enter One step improves the efficiency that screening is susceptible to suffer from the biological sample of cone rod cell muscular dystrophy.According to embodiments of the invention, here The term " sample of nucleic acid " used should be interpreted broadly, and it can be any can to reflect in biological sample, whether CRX exists The sample of sudden change, such as, can be the complete genome DNA of extracting directly from biological sample, it is also possible to is to wrap in this full-length genome Containing a part for CRX coded sequence, can be the total serum IgE extracted from biological sample, it is also possible to be to extract from biological sample MRNA.According to one embodiment of present invention, described sample of nucleic acid is complete genome DNA.Thus, it is possible to expand biology Sample carry out source range, and can the much information of biological sample be determined such that it is able to improve screening and be susceptible to suffer from regarding simultaneously The efficiency of the biological sample of cone rod cell muscular dystrophy.It addition, according to embodiments of the invention, the type of sample of nucleic acid is also Being not particularly limited, for using RNA as sample of nucleic acid, then nucleic acid-extracting apparatus farther includes RNA extraction unit 101 With reverse transcription unit 102, wherein, extraction unit 101 is for from extraction from biological material RNA sample, reverse transcription unit 102 and RNA Extraction unit 101 is connected, for RNA sample is carried out reverse transcription reaction, in order to obtain cDNA sample, obtained cDNA Sample constitutes sample of nucleic acid.RNA is utilized to be susceptible to suffer from cone rod cell battalion as sample of nucleic acid screening thus, it is possible to improve further Support the efficiency of the biological sample of bad disease.

According to embodiments of the invention, nucleotide sequence determines that device 200 is connected with nucleic acid-extracting apparatus 100, for sample of nucleic acid It is analyzed, in order to determine the nucleotide sequence of sample of nucleic acid.According to embodiments of the invention, determine the core of obtained sample of nucleic acid The method and apparatus of acid sequence is not particularly restricted.According to a particular embodiment of the invention, the method for order-checking can be used to determine The nucleotide sequence of sample of nucleic acid.Thus, according to one embodiment of present invention, described nucleotide sequence determines that device 200 can enter One step includes: library construction unit 201 and order-checking unit 202.Library construction unit 201, for for sample of nucleic acid, builds Nucleic acid sequencing library；Order-checking unit 202 is connected with library construction unit 201, for checking order nucleic acid sequencing library, in order to Obtain the sequencing result being made up of multiple sequencing datas.

About for sample of nucleic acid, building method and the flow process of sequencing library, those skilled in the art can be according to different order-checkings Platform suitably selects, and about the details of flow process, the such as Illumina company of manufacturer of the instrument that may refer to check order is provided Code, for example, see Illumina company Multiplexing Sample Preparation Guide (Part#1005361；Feb 2010) Or Paired-End SamplePrep Guide (Part#1005063；Feb 2010), by referring to being incorporated into herein.According to this Inventive embodiment, from the method and apparatus of extraction from biological material sample of nucleic acid, is also not particularly limited, and can use commercialization Nucleic acid extraction kit carry out.

It should be noted that the term here used " nucleotide sequence " should broadly understood, it can be to nucleic acid sample Originally the complete nucleic acid sequence information obtained after the sequencing data obtained assembles that checks order is carried out, it is also possible to be directly to use to pass through The sequencing data (reads) checking order obtained to sample of nucleic acid is as nucleotide sequence, as long as containing right in these nucleotide sequences Answer the coded sequence of CRX.

It addition, according to embodiments of the invention, can screen sample of nucleic acid, being enriched with CRX exon, this screening is enriched with Can be before building sequencing library, during building sequencing library, or carry out after structure sequencing library.Thus, library Construction unit 201 may further include PCR and expands module (not shown), is provided with CRX in this PCR amplification module Exon specific primer, in order to utilize CRX exon specific primer, described sample of nucleic acid is carried out PCR amplification.Thus, Can be expanded by PCR, be enriched with CRX exon such that it is able to improve screening further and be susceptible to suffer from cone rod cell malnutrition The efficiency of the biological sample of disease.According to a particular embodiment of the invention, CRX exon specific primer has such as SEQ ID NO: Nucleotide sequence shown in 3 and 4:

Forward primer: 5 '-CCTCTGGGCATCTCAGAT-3 ' (SEQ ID NO:3)；

Reverse primer: 5 '-AGGAAAGGGGTGGTCTCT-3 ' (SEQ ID NO:4).

It is surprisingly found by the inventors that, by using above-mentioned primer, significantly can effectively complete CRX in PCR reaction system The amplification of exon.It should be noted that the nucleotide sequence shown in these SEQ ID NO:3 and SEQ ID NO:4 is this The inventor of invention, after having paid arduous labor, surprisingly obtains.

According to embodiments of the invention, the equipment that may be used for carrying out checking order is not particularly restricted.According to embodiments of the invention, Second filial generation order-checking platform can be used, it would however also be possible to employ the order-checking platform of the third generation and forth generation or more advanced.According to this Bright concrete example, order-checking unit 202 can be selected from HISEQ2000, SOLiD, 454 and single-molecule sequencing device at least A kind of.Thus, in conjunction with up-to-date sequencing technologies, the higher order-checking degree of depth can be reached for Single locus, detection sensitivity and Accuracy is greatly improved, it is thus possible to utilize the feature that the high flux of these sequencing devices, the degree of depth check order, and improves core further Acid sample carries out the efficiency that detection is analyzed.Thus, improve follow-up accuracy time sequencing data is analyzed and accuracy.

According to embodiments of the invention, it is judged that device 300 determines that with nucleotide sequence device 200 is connected, and is suitable to the core of sample of nucleic acid Acid sequence is compared, in order to whether nucleotide sequence based on sample of nucleic acid judges biological sample with the difference of SEQ ID NO:1 It is susceptible to suffer from cone rod cell muscular dystrophy.Thus, this system is utilized, it is possible to screening is susceptible to suffer from cone rod cell nutrition effectively The biological sample of bad disease.

Specifically, nucleotide sequence based on sample of nucleic acid is compared with SEQ ID NO:1, if having c.766C > T sudden change, sentence Whether disconnected biological sample is susceptible to suffer from cone rod cell muscular dystrophy.As it was previously stated, according to one embodiment of present invention, nucleic acid C.766C the nucleotide sequence of sample, compared with SEQ ID NO:1, has > T sudden change is that biological sample is susceptible to suffer from cone rod cell The instruction of muscular dystrophy.According to embodiments of the invention, the equipment comparing nucleotide sequence and SEQ ID NO:1 is also It is not particularly limited, the software of any conventional can be used to operate, according to the instantiation of the present invention, SOAP can be used Software is compared.

According to the fourth aspect of the invention, the present invention proposes a kind of life being susceptible to suffer from cone rod cell muscular dystrophy for screening The test kit of thing sample.According to embodiments of the invention, this is used for screening the biological sample being susceptible to suffer from cone rod cell muscular dystrophy The test kit of product includes: be adapted to detect for the reagent of CRX gene mutation body, wherein compared with SEQ ID NO:1, and this CRX gene C.766C mutant has > T sudden change.Utilize test kit according to an embodiment of the invention, it is possible to screening is susceptible to suffer from the cone and regards effectively The biological sample of rod cell muscular dystrophy.In this article, the term used " is adapted to detect for the reagent of CRX gene mutation body " Should be interpreted broadly, can be i.e. the reagent of detection CRX encoding gene, it is also possible to be the reagent of detection CRX mutant polypeptide, Such as can use the antibody in identification specificity site.According to one embodiment of present invention, described reagent is nucleic probe or draws Thing, it is preferable that described nucleic probe or primer have the nucleotide sequence as shown in SEQ ID NO:3-4.Thus, it is possible to Screening efficiently is susceptible to suffer from the biological sample of cone rod cell muscular dystrophy.

It should be noted that in the components of system as directed of the biological sample that screening is susceptible to suffer from cone rod cell muscular dystrophy herein above Described feature and advantage, are equally applicable to screen the test kit of the biological sample being susceptible to suffer from cone rod cell muscular dystrophy, Do not repeat them here.

Construct and reconstitution cell

According to the fifth aspect of the invention, the invention allows for a kind of construct.According to embodiments of the invention, this construct Comprise the nucleic acid of the coding CRX mutant of foregoing separation, i.e. the CRX gene mutation body of the present invention.Thus, this is utilized The reconstitution cell that the construct transformed acceptor cell of invention obtains, it is possible to be efficiently used for screening treatment cone rod cell nutrition not The medicine of good disease.Wherein, the kind of described recipient cell is not particularly limited, can be such as Bacillus coli cells, suckling move Thing cell, preferably this receptor cell derived is in mammal.

The term " construct " used in the present invention refers to such a kind of heredity carrier, and it comprises specific nucleic acid sequence, And can purpose nucleotide sequence be proceeded in host cell, to obtain reconstitution cell.According to embodiments of the invention, construct Form be not particularly limited.According to embodiments of the invention, it can be plasmid, phage, artificial chromosome, cosmid (Cosmid), virus at least one, preferred plasmid.Plasmid, as heredity carrier, has simple to operate, can carry relatively The character of large fragment, it is simple to operate and process.The form of plasmid is also not particularly limited, and both can be circular plasmids, it is also possible to It is linear plasmid, can be i.e. strand, it is also possible to be double-strand.Those skilled in the art can select as required. The term " nucleic acid " used in the present invention can be any polymer comprising deoxyribonucleotide or ribonucleotide, Include but not limited to that its length is the most any particular limitation through that modify or the most modified DNA, RNA.For with In the construct of structure reconstitution cell, the most described nucleic acid is DNA, because DNA is for RNA, it is more stable, And it is easily operated.

According to the sixth aspect of the invention, the invention allows for a kind of reconstitution cell.According to embodiments of the invention, this restructuring Cell is obtained by foregoing construct transformed acceptor cell.Thus, the reconstitution cell of the present invention can express structure Build the CRX gene mutation body entrained by body.According to some embodiments of the present invention, utilize the reconstitution cell of the present invention, it is possible to have The medicine of effect ground screening treatment cone rod cell muscular dystrophy.According to embodiments of the invention, the kind of recipient cell is not subject to Limiting especially, such as, can be Bacillus coli cells, mammalian cell, the most described recipient cell derives from inhuman suckling and moves Thing.

The method building medicaments sifting model

According to the seventh aspect of the invention, present invention also offers a kind of method building medicaments sifting model.According to the present invention's Embodiment, the method includes: make at least some of cell of animal express the nucleic acid of aforesaid coding CRX mutant.According to this Inventive embodiment, described animal is mice, pig, Canis familiaris L., primate.According to some embodiments of the present invention, utilize this The medicaments sifting model of invention, it is possible to the medicine of screening treatment cone rod cell muscular dystrophy effectively.

It should be noted that the method that the present invention builds medicaments sifting model is not particularly limited, as long as making at least the one of animal Part cell expresses aforesaid CRX gene mutation body.For example, it is possible to by the method using gene transformation, by noted earlier The construct of the present invention proceed to receptor (inhuman), so that at least some of cell reaching animal expresses aforesaid CRX Gene mutation body；Marker-free transgenic technology, CRISPR/Cas9 gene editing technology, site-directed integration technology etc. can also be used, C.766C the CRX gene making receptor occurs > T sudden change, and the aforesaid polypeptide of effective expression, thus this receptor animal can send out Raw cone rod cell muscular dystrophy, and then the medicine of screening treatment cone rod cell muscular dystrophy can be efficiently used for Thing, namely medicaments sifting model can be acted effectively as.

It should be noted that the present invention uses the full exon group sequencing technologies of a new generation, for an ADCORD patient home System carries out full-length genome exon group sequencing analysis, thus is found that a Disease-causing gene CRX new for ADCORD, and this base Because of upper pathogenic mutation.Thus, the present invention enriches Disease-causing gene and the pathogenic mutation collection of illustrative plates of CRX gene, further illustrates The Molecular pathogenesis of cone rod cell muscular dystrophy, the early stage Disease-causing gene for cone rod cell muscular dystrophy 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 merely illustrative, And be not considered as limiting the invention.

If not specializing, the conventional means that the technological means employed in embodiment is well known to those skilled in the art, permissible Carrying out with reference to " Molecular Cloning: A Laboratory guide " third edition or Related product, the reagent used and product are also and can business obtain ?.The various processes not described in detail and method are conventional methods as known in the art, the source of agents useful for same, trade name And be necessary to list its constituent person, all indicate when occurring first, identical reagent used is if no special instructions thereafter, all Identical with the content indicated first.

Embodiment 1 determines cone rod cell muscular dystrophy pathogenic mutation

1, sample collection

Inventor one occupy the ill family of adCORD of southern china, and its pedigree chart is shown in Fig. 2.As in figure 2 it is shown, wherein, zero Representing normal female, represents that normal male, ■ represent male patient, ● represent female patient,Represent late male Patient；Represent late normal male；Represent late normal female；Arrow indication is proband (III-12).

This family is existing 9 patients, continuous 3 generations morbidity, and men and women all gets involved, and meets Mendel's autosomal dominant and loses Arq mode.The proband (III-12) of this family is male, 50 years old.It is diagnosed as gradual visual deterioration during proband 40 years old With slight color defect, fundus photography (fundus photography) finds that this patient's macula lutea has 2 × 2PD native gold sample Reflection, diffusivity hypopigmentation；Peripherality retinal manifestations goes out spicule shape pigmentation and arteria retina decay, sees figure 3A-Fig. 3 B；Optical coherence tomography (optical coherence tomography, OCT) checks and finds, its macular area regards Nethike embrane is thinning, sees Fig. 3 C-Fig. 3 D；Fundus autofluorescence (fundus autofluorescence) finds, macular area has the most glimmering Light, but have no choroidal atrophy around, see Fig. 3 E-Fig. 3 G；Retinal current traces (electroretinography, ERG) Find that the cone responses of proband is the slowest, and rod cell reaction is normal.Other patient's phenotypes of this family are similar.

Inventor acquires the peripheral blood of all members alive in this family.According to world medicine united organization, " Helsinki is declared Speech " requirement of (Declaration of Helsinki), experimenter endorsed relevant Informed Consent Form to Hospital Ethical Committee.

2, target area capture order-checking

Inventor utilize Aglient SureSelect Human All Exon kit (Agilent Technologies, Santa Clara, CA, USA) Solexa high throughput sequencing technologies is combined, to 3 trouble in above-mentioned cone rod cell muscular dystrophy patient's family The exon group sequence of person (III-4, III-12 and IV-7) and 1 normal person (III-10) is checked order, wherein with 100 The outer normal person of name family is as comparison.

Specific as follows:

2.1 DNA extraction

Under aseptic condition, extract this family member III-4 (patient), III-10 (normal person), III-12 (proband) and IV-7 The periphery anticoagulation of (patient), uses peripheral blood DNA purification kit (Qiagen company of Germany) to carry out genome respectively The extraction of DNA and purification, experimental procedure is carried out according to QIAGEN description.

The quality testing of DNA sample: with ultra-pure water school zero Nanodrop ultramicrospectrophotometer, measure the DNA of extracting Sample light absorption value and the ratio of sample light absorption value under 280nm ultraviolet light under the concentration of sample and 260nm ultraviolet light.Gained every The OD of individual sample genomic dna₂₆₀/OD₂₈₀All should be between 1.7-2.0, concentration is no less than 200ng/ μ l, and total amount is many In 30 μ g.

2.2 target area captures and order-checking

Ultrasonoscope (CovarisS2, Massachusetts, USA) is utilized to be broken at random by each genomic DNA sample The fragment of about 150-200bp, the operating instruction provided according to manufacturer subsequently, connect top connection system respectively at fragment two ends Standby library (can be found in: the Illumina/Solexa standard that http://www.illumina.com/ provides builds storehouse description, by ginseng According to being incorporated by herein).I.e. be available on the machine after library detection is qualified order-checking, in order to obtains raw sequencing data.Wherein, ginseng Checking order according to the cluster of Illumina standard and the step of order-checking, order-checking platform is Illumina Hiseq 2000, reads length For 90bp, both-end checks order.Wherein, the full exon group sequence of 4 example experimenters (III-4, III-10, III-12 and IV-7) is flat All the order-checking degree of depth be respectively 86.48 ×, 100.39 ×, 97.03 × and 94.38 ×.

3, variation detection, annotation and data base compare

Utilize Illumina basecalling Software 1.7 that the raw sequencing data of above-mentioned acquisition is processed, filter off through crossing After pollution, SOAPaligner/SOAP2 is used (to can be found in: 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 ultrafast tool for short read alignment.Bioinformatics 2009, 25 (15): 1966-1967, by referring to being incorporated by herein) comparison to reference genome UCSC NCBI37/hg19, with Just the comparison unique aligned sequences to genome is obtained.Then SOAPsnp is utilized (to can be found in: Li R, Li Y, Fang X, Yang H,et al,SNP detection for massively parallel whole-genome resequencing.Genome Res 2009, 19 (6): 1124-1132, by referring to being incorporated by herein) determine the genotype of target region.

BWA (version 0.7.5) is used (to see Li H, Durbin R.Fast and accurate long-read alignment with Burrows-Wheeler transform.Bioinformatics 2010,26 (5): 589-595, by referring to being incorporated by herein) (DePristo MA, Banks E, Poplin R, et is seen with GATK (Genome Analysis Toolkit) (version2.8-1) al.A framework for variation discovery and genotyping using next-generation DNA sequencing Data.Nat Genet 2011,43:491-498, by referring to being incorporated by herein) determine Indel (insertion-deletion, Insertion/deletion labelling) type.

As a result, inventor finds 109560 single nucleotide polymorphism (SNP) in III-4, finds 108 in III-10 907 single nucleotide polymorphism (SNP), find 110292 single nucleotide polymorphism (SNP), at IV-in III-12 108350 single nucleotide polymorphism (SNP) are found in 7.

Subsequently by dbSNP data base (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi), thousand Human genome data base (http://www.1000genomes.org/) and HapMap data base (http://hapmap.ncbi.nlm. Nih.gov/) filtration of public database, removes all known and be more than 0.005 in data base's allelic frequency variation (variation MAF value is high, the most non-pathogenic common polymorphism).And then, for reducing the hunting zone of Disease-causing gene sudden change, Sudden change, intragenic mutation and samesense mutation between gene are removed by inventor, lack non-synonym/splice site sudden change and small insertion Mistake carries out prioritizing selection.By comparing 3 example patients (III-4, III-12, IV-7) and the change of 1 example normal individual (III-10) Different, find that 3 example patients all carry 26 identical mutant genes.Further, inventor utilize forecasting software SIFT to this 2 6 sudden changes have carried out hazardness prediction, finally think: CRX gene is the Disease-causing gene of this adCORD family.Specifically, For this adCORD family, CRX gene there occurs nonsense mutation: c.766C > T (p.Q256X), causes polypeptide chain to be translated Termination.

Known CRX gene is positioned at No. 19 chromosome long arm 1 district 3 of people and carries (19q13), containing 5 exons, encodes 299 Individual amino acid residue.CRX gene is expressed at amphiblestroid inner nuclear layer, by with other transcription factor (such as NRL and RX) Synergism come differentiation and the maintenance of sense of participation photo-cell.CRX protein molecular has a similar paired homology segment, One WPS domain, and it is positioned at the OTX tail of c-terminus.OTX tail is that CRX plays regulation and control as transcription factor The important feature of effect.CRX forms dimer by OTX tail and neural retina leucine zipper (NRL) and plays alive Property.Thus, OTX tail is extremely important to CRX gene.Inventor finds, sudden change is (as c.504delA, c.587delCC CC, c.816delCACinsAA and c.C766T) may result in OTX tail disappear, affect the expression of photoreceptor cell，photosensory cell specific gene, Because OTX tail can set up the interaction of CRX and other transcription factor.Though lacking CTX albumen and the mesh of OTX tail Mark promoter combines, but does not interacts, thus may hinder the transcription activating of photoreceptor cell，photosensory cell specific gene, and then causes adCORD。

Further, Sanger verifies discovery, and CRX gene is c.766C > T sudden change exist only in this adCORD patient's family In patient, and in family, the outer normal control individuals of the family of normal individual and 100 example affinity-less relations all suddenlys change without this.

Thus, it has been recognised by the inventors that the Disease-causing gene that CRX gene is this adCORD patient's family, CRX gene is c.766C > T (p.Q256X) sudden change, for the pathogenic mutation of cone rod cell muscular dystrophy.

Embodiment 2 Sanger method sequence verification

Respectively to all family members (including patient and normal family members) in the adCORD patient's family described in embodiment 1, And the CRX gene of 100 outer normal persons of family detects: for CRX gene c.766C > T sudden change design primer, Then expanded by PCR, product purification obtains the relevant sequence in mutational site with the method for order-checking, according to determining that sequencing is tied Fruit belongs to saltant type or wild type, relevant between checking CRX gene and this sudden change to cone rod cell muscular dystrophy Property.

Concrete grammar step is as follows:

1, DNA extraction

According to the method for the extraction DNA described in embodiment 1, extract the genome in preparation experimenter's peripheric venous blood respectively DNA, standby.

2, design of primers and PCR reaction

First, reference man genoid data unit sequence storehouse GRCh37/hg19, design obtains having shown in SEQ ID NO:3-4 The CRX gene extron specific primer of nucleotide sequence, particular sequence is as follows:

Forward primer: 5 '-CCTCTGGGCATCTCAGAT-3 ' (SEQ ID NO:3)；

Reverse primer: 5 '-AGGAAAGGGGTGGTCTCT-3 ' (SEQ ID NO:4).

Then, respectively according to the PCR reaction system of the following proportioning each genomic DNA sample of preparation:

Reaction system (25 μ l):

Then, each PCR reaction system is carried out respectively PCR reaction according to following reaction condition:

95 DEG C 2 minutes；35 circulations: (94 DEG C minutes, 62 DEG C 30 seconds, 72 DEG C 30 seconds)；72 DEG C, 7 minutes.

Thus, it is thus achieved that each receptor gene organizes the pcr amplification product of DNA sample.

3, order-checking

The pcr amplification product that each receptor gene obtained in step 2 organizes DNA sample is directly carried out DNA sequencing.Its In, order-checking uses Genetic Analyzer 3500 sequenator to carry out.

Based on sequencing result, this sudden change to CRX gene in cone rod cell muscular dystrophy patient's family of the present invention Site carries out sudden change investigation, found that c.766C all existing cases all carry in this family > heterozygous mutant of T (p.Q256X), And its normal family members and the outer normal population of 100 example familys the most do not carry this sudden change.Wherein, Fig. 4 shows above-mentioned cone retinal rod Healthy member in proband and family in cytotrophy bad disease patient's family, and the CRX gene of the outer normal person of family C.766C > the representative Sanger sequence verification peak figure in T mutational site.

Thus, prove the Disease-causing gene that CRX gene is cone rod cell muscular dystrophy further, CRX gene C.766C > T sports the pathogenic mutation of this disease.

Embodiment 3 detection kit

Preparing a detection kit, c.766C it comprises can detect CRX gene > T sudden change primer, be susceptible to suffer from for screening The biological sample of cone rod cell muscular dystrophy, wherein these primers are CRX gene extron specific primer, its sequence Arrange as described in example 2 above shown in SEQ ID NO:3-4.

The screening of mentioned reagent box is utilized to be susceptible to suffer from the concretely comprising the following steps of biological sample of cone rod cell muscular dystrophy: according to enforcement Method described in 2.1 " DNA extraction " of example 1 step 2 extracts person DNA to be measured, with the DNA that extracted for template with The exon specific primer of above-mentioned CRX gene carries out PCR reaction (PCR reaction system and reaction condition see embodiment 2), And according to this area conventional method to PCR primer purification, the product of purification is checked order, then by observing order-checking gained To sequence whether have c.766C > T sudden change, it is possible to effectively detect the CRX gene mutation body of the present invention at person DNA to be measured In whether exist such that it is able to effectively detect whether person to be measured is susceptible to suffer from cone rod cell muscular dystrophy, further, it is possible to The biological sample being susceptible to suffer from cone rod cell muscular dystrophy is filtered out from person to be measured.

In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " concrete example ", Or specific features, structure, material or the feature bag that the description of " some examples " etc. means to combine this embodiment or example describes It is contained at least one embodiment or the example of the present invention.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 at any one Or multiple embodiment 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: without departing from this These embodiments can be carried out multiple change in the case of the principle of invention and objective, revise, replace and modification, the present invention's Scope is limited by claim and equivalent thereof.

Claims (10)

1. the nucleic acid of coding CRX mutant separated, it is characterised in that described nucleic acid compared with SEQ ID NO:1, Have c.766C T sudden change,

Optionally, described nucleic acid is DNA.

2. the polypeptide separated, it is characterised in that compared with SEQ ID NO:2, the polypeptide of described separation has p.Q256X Sudden change,

Optionally, described polypeptide is by the nucleic acid coding described in claim 1.

3. a screening is susceptible to suffer from the system of biological sample of cone rod cell muscular dystrophy, it is characterised in that including:

Nucleic acid-extracting apparatus, described nucleic acid-extracting apparatus is for from described extraction from biological material sample of nucleic acid；

Nucleotide sequence determines that device, described nucleotide sequence determine that device is connected with described nucleic acid-extracting apparatus, for described nucleic acid Sample is analyzed, in order to determine the nucleotide sequence of described sample of nucleic acid；

Judgment means, with described nucleotide sequence, described judgment means determines that device is connected, in order to nucleic acid based on described sample of nucleic acid Sequence or its complementary series, compared with SEQ ID NO:1, if having c.766C > T sudden change, it is judged that described biological sample Whether it is susceptible to suffer from cone rod cell muscular dystrophy.

System the most according to claim 3, it is characterised in that described nucleic acid-extracting apparatus farther includes:

RNA extraction unit, described RNA extraction unit is for from described extraction from biological material RNA sample；And

Reverse transcription unit, described reverse transcription unit is connected with described RNA extraction unit, for carrying out described RNA sample Reverse transcription reaction, in order to obtain cDNA sample, described cDNA sample constitutes described sample of nucleic acid.

System the most according to claim 3, it is characterised in that described nucleotide sequence determines that device farther includes:

Library construction unit, described library construction unit, for for described sample of nucleic acid, builds nucleic acid sequencing library；And

Order-checking unit, described order-checking unit is connected with described library construction unit, for being checked order in described nucleic acid sequencing library, To obtain the sequencing result being made up of multiple sequencing datas.

System the most according to claim 5, it is characterised in that described library construction unit farther includes:

PCR expands module, is provided with CRX gene extron specific primer, in order to utilize institute in described PCR amplification module State specific primer, described sample of nucleic acid carried out PCR amplification,

Optionally, described specific primer has the nucleotide sequence as shown in SEQ ID NO:3-4,

Optionally, described order-checking unit includes selected from HISEQ2000, SOLiD, 454 and at least the one of single-molecule sequencing device Kind.

7. the test kit of the biological sample being susceptible to suffer from cone rod cell muscular dystrophy for screening, it is characterised in that contain Have:

It is adapted to detect for the reagent of CRX gene mutation body, wherein compared with SEQ ID NO:1, described CRX gene mutation body Have c.766C T sudden change,

Optionally, described reagent is nucleic probe or primer,

Optionally, described nucleic probe or primer have the nucleotide sequence as shown in SEQ ID NO:3-4.

8. a construct, it is characterised in that comprise the nucleic acid of the coding CRX mutant of separation described in claim 1.

9. a reconstitution cell, it is characterised in that described reconstitution cell is to be converted by the construct described in claim 8 to be subject to Somatic cell and obtain.

10. the method building medicaments sifting model, it is characterised in that including:

At least some of cell making animal expresses the nucleic acid described in claim 1,

Optionally, described animal is mice, pig, Canis familiaris L., primate.