CN105624167B - A kind of pathogenic mutation and its detection reagent of heredity centrality halo shape retinopathy - Google Patents

A kind of pathogenic mutation and its detection reagent of heredity centrality halo shape retinopathy Download PDF

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CN105624167B
CN105624167B CN201610076303.0A CN201610076303A CN105624167B CN 105624167 B CN105624167 B CN 105624167B CN 201610076303 A CN201610076303 A CN 201610076303A CN 105624167 B CN105624167 B CN 105624167B
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guca1a
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mutation
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赵晨
陈雪
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Jiangsu Province Hospital First Affiliated Hospital With Nanjing Medical University
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/164—Retinal disorders, e.g. retinopathy

Abstract

The invention discloses the pathogenic mutations and its detection reagent of a kind of heredity centrality halo shape retinopathy.It is a kind of for detecting the GUCA1A gene of heredity centrality halo shape retinopathy, the GUCA1A gene of mutation is heterozygous mutant GUCA1A p.Arg120Leu.A kind of kit detecting heredity centrality halo shape retinopathy, the kit include: the reagent that (a) detection GUCA1A gene physical location is 42146547 and 42146548 nucleotide sites；Or the reagent in detection the 120th amino acids site of 1 albumen of GCAP；(b) specification.The present invention obtains the mutation GUCA1A gene of heredity centrality halo shape retinopathy, can carry out the diagnosis of heredity centrality halo shape retinopathy by detecting the mutation.

Description

A kind of pathogenic mutation of heredity centrality halo shape retinopathy and its detection Reagent
Technical field
The invention belongs to biomedicine fields, are related to a kind of pathogenic mutation of heredity centrality halo shape retinopathy And its detection reagent.
Background technique
Centrality halo shape retinopathy (central areolar choroidal dystrophy；It CACD) is one Group progressive retina degenerative disease as caused by genetic defect, this kind of disease with retinal pigment epithelium (RPE) and The irreversibility atrophy of choroidal artery is major pathologic features.The macular area of CACD patient main harm patient, causes center The progressive of eyesight declines, and serious person can blinding.CACD is a kind of heredity blinding clinically common and that harm is more serious Disease can be divided into four phases by its Clinical symptoms.Early stage CACD patient since its phenotype is easily mutually obscured with other eyeground patient, Therefore it is difficult to make it accurate clinical diagnosis.Therefore, other the diagnosis sides that can potentially assist CACD clinical diagnosis are found Formula is particularly important.
CACD is single gene inheritance disease, has significant genetic heterogeneity.The hereditary pattern being currently known is autosome Dominant inheritance, it is known that Disease-causing gene there are two, be PRPH2 and GUCY2D.However, the mutation of the two genes can not solve The inherited pathogenic factor of whole CACD patients is released, still has the Disease-causing gene of a large amount of CACD patients not yet to find, there are unknown for prompt The new Disease-causing gene of CACD has to be excavated.It must be set up for the molecule genetics research of CACD in certain molecular biology skill On the basis of art.One free-revving engine of research CACD Disease-causing gene is the molecular diagnosis for carrying out CACD, heterogeneous in view of its heredity Property, the research method for seeking a kind of completely new CACD Disease-causing gene seems especially urgent.High-throughput two generation sequencing technologies can be high The hereditary information for lots of genes of effect is captured, and has the untouchable advantage of traditional technology institute.
Guanylyl cyclase-activating protein 1(GUCA1A；MIM 600364) gene be located at No. 6 dye The position colour solid galianconism 6p21.1, the gene contain 6 exons, and 1 albumen of GCAP of coding is that one kind contains 201 amino The retinal specific of acid expresses albumen, and research has been reported that GUCA1A gene mutation can cause cone cell degeneration and cone at present Rod cell is malnutritive, but its relationship between CACD is showed no any report both at home and abroad.
Summary of the invention
The purpose of the present invention is in view of the foregoing drawbacks, provide a kind of causing a disease for heredity centrality halo shape retinopathy Mutation.
It is a further object of the present invention to provide the applications of the pathogenic mutation.
It is yet another object of the invention to provide the detection reagents of the pathogenic mutation.
The purpose of the present invention can be achieved through the following technical solutions:
It is a kind of for detecting the GUCA1A gene of heredity centrality halo shape retinopathy, the GUCA1A gene of mutation For heterozygous mutant GUCA1A p.Arg120Leu.
Gene number of the wild type GUCA1A gene in Ensemble database are as follows: ENSG00000048545, it is described The GUCA1A gene of mutation sports T by G in the base that physical location is 42146547, the alkali for being 42146548 in physical location Base sports T by C, and other parts are identical as wild type.
A kind of 1 albumen of GCAP of mutation, gene transcripts of 1 albumen of wild type GCAP in Ensemble database are compiled Number are as follows: 1 albumen of GCAP of ENST00000394237, mutation are become in the 120th amino acids of the wild-type protein by arginine For leucine, other parts are identical as wild type.
The GUCA1A gene of mutation of the present invention or 1 albumen of GCAP of the mutation are in preparation heredity Application in disposition halo shape retinopathy detection reagent or detection device.
Wherein the detection reagent preferably is selected from: one of primer or primer pair, probe, antibody or nucleic acid chip or It is a variety of.
The detection device preferably includes the detection platform of the genetic chip of the GUCA1A gene containing detection mutation.
A kind of kit detecting heredity centrality halo shape retinopathy, the kit include:
(a) detection GUCA1A gene physical location is the reagent of 42146547 and 42146548 nucleotide sites；Or detection The reagent in the 120th amino acids site of 1 albumen of GCAP；
(b) specification.
Wherein, the reagent is preferably selected from one of primer or primer pair, probe, antibody or nucleic acid chip or more Kind.
Preferably as one kind of the invention, the reagent is to be visited based on the gene chip hybridization that deep sequencing is platform Needle.
The hybridization probe sequence for detecting 42146547 and 42146548 nucleotide sites is preferably chr6 | 42146509- 42146628, sequence is as shown in SEQ ID NO.14.
Preferably as another kind of the invention, the reagent is 42146547 and 42146548 nucleotide sites of detection Primer pair.
The forward primer sequence for detecting 42146547 and 42146548 nucleotide sites is SEQ ID NO.17, reverse primer Sequence is SEQ ID NO.18.
A method of using deep sequencing as GUCA1A gene mutation in platform screening CACD patient, comprising the following steps:
(1) CACD patient's family clinic and genetic resource are established, the clinical data and blood preparation of RP family is collected, mentions Take genomic DNA；
(2) it designs and is visited shown in SEQ ID NO.1~SEQ ID NO.16 for detecting the hybridization of GUCA1A gene mutation Needle, and be integrated on genetic chip；
(3) target area is captured using the genetic chip of preparation and carry out deep sequencing；
(4) bioinformatic analysis optimized to sequencing result, the pathogenic mutation for screening a new CACD are Heterozygous mutant GUCA1A p.Arg120Leu.Mutation GUCA1A p.Arg120Leu is located at No. 6 chromosomes, and physical location is The base of 42146547 and 42146548 (Ensemble databases) sports TT by GC；Rna level: GUCA1A gene coding RNA the 359th and 360 bit bases sport TT by GC；Protein level: the 120th amino acids of SPP2 gene coded protein are by smart ammonia Acid becomes leucine.
The genetic chip of the preferred Agilent company production of genetic chip described in step (2).
Genetic chip described in step (3) using preparation captures target area and carries out deep sequencing preferably by the U.S. The Hi-seq2000 instrument of Illumina company is completed.
Genetic chip described in step (3) using preparation captures target area and carries out deep sequencing preferred flow are as follows: By genomic DNA fragment, it is connected in DNA end mark " A " and with Illumina PE connector-oligonucleotide mixture；Even Object of practicing midwifery is enriched with through PCR, obtains DNA library, and DNA library and known Disease-causing gene are captured chip hybridization, elution, purifying, Obtain coded sequence；Creation pairing end, is sequenced target sequence on 2000 platform of Illumina HiSeqTM.
Beneficial effect
1.CACD is common, serious heredity blinding disease, and in China, disease incidence is higher, has been seriously endangered national strong Health.The new pathogenic mutation and new Disease-causing gene for excavating CACD are conducive to the Molecular etiology for further exploring CACD, are to fill Divide the ophthalmology hereditary disease resource using China, benefits the real needs of CACD patient, be genome times afterwards comprehensively most important research One of direction.This patent is intended to explore the genetic factors of CACD, to help to understand pathogenesis, adjuvant clinical diagnosis, produce Preceding diagnosis and therapeutic transgene.
2. at present not some studies pointed out that the relationship between GUCA1A gene mutation and centrality halo shape retinopathy, because This, the correlative study for GUCA1A gene and CACD seems particularly necessary.This patent selects GUCA1A gene as CACD's Candidate gene is to be filtered out on the basis of being engaged in clinical treatment, genetics research for many years by applicant, through the invention Method has further clarified GUCA1A gene and the relationship of CACD.
3. a gene can correspond to multiple and different transcripts, and the encoded RNA of different transcripts and albumen are It is different.Applicant filters out the optimal transcript of GUCA1A gene according to the experience of long campaigns genetics research in this patent, And Inherited retinal is finally obtained so that screening benefit be made to reach highest according to the different corresponding probes of transcription the design The pathogenic mutation GUCA1A p.Arg120Leu of pigment denaturation.
4.CACD has certain genetic heterogeneity, and the Disease-causing gene being currently known includes PRPH2 and GUCY2D, but still is deposited In unknown Disease-causing gene.The present invention provides the pathogenic sites of new Disease-causing gene, for the disease diagnosis provide it is new Molecular biology mechanism.
Detailed description of the invention
Fig. 1: pedigree chart
Fig. 2: CACD1 phase patient eyeground photograph, fundus autofluorescence, fluorescence fundus angiography (patient IV:10)
Fig. 3: CACD2 phase patient eyeground photograph, fundus autofluorescence, fluorescence fundus angiography (patient III:16)
Fig. 4: CACD3 phase patient eyeground photograph, fundus autofluorescence, fluorescence fundus angiography (patient III:14)
Fig. 5: CACD4 phase patient eyeground photograph, fundus autofluorescence, fluorescence fundus angiography (patient III:18)
Fig. 6: GUCA1A mutation and wild-type sequence sequencer map
Fig. 7: conservative Analysis
Fig. 8: wild type and the GUCA1A albumin crystal structure for carrying mutation
Fig. 9: zebra fish configuration structure
Figure 10: zebra fish eye morphosis
Figure 11: zebra fish eyeball is sliced immunofluorescence dyeing
Figure 12: zebra fish choroidal artery morphosis
Specific embodiment
Embodiment 1
To a five generation centrality halo shape retinopathy (central areolar choroidal dystrophy； CACD the GUCA1A gene mutation of family) is detected.
Experimental method:
1. the acquisition of the family clinical resources and the foundation of genetic resource:
The clinical data and blood sample of each member in the family are collected, pedigree chart is shown in Fig. 1.Clinical data mainly includes People's medical history, family history, most preferably correct defects of vision (best corrected visual acuities；BCVAs), slit lamp examination, Eyeground photograph, chromatoptometry, perimetry (visual field Humphrey meter), visual evoked potential detect (visual-evoked potentials；VEP), full visual field electrophysiologic study (electroretinography；ERG), fluorescence fundus angiography is examined Look into (fundus fluoresceinangiography；) and optical coherence tomography inspection (optical coherence FFA tomography；OCT) etc..And it is each to family with poba gene group DNA extraction kit (Qiagen, Hilden, Germany) The poba gene group DNA of member is extracted.
2. being excavated the pathogenic mutation of the family by means of high-throughput two generations sequencing:
2.1 designs and customized capture chip:
2.1.1SPP2 gene and transcript sequence information:
The gene trap chip is that full exon group captures chip, can be detected to current all knowns, wherein The relevant Disease-causing gene of CACD including being all currently known.The GUCA1A gene of our institute's references is in Ensemble database Gene number are as follows: ENSG00000048545 (note: the number come from Ensemble database,www.ensembl.org, can be defeated Enter gene coding retrieval gene details and gene order).
2.1.2 the selection of transcript:
Specific transcript is selected for different genes, each gene contains multiple transcripts, when selecting transcript, Our principle is: the transcript for possessing CCDS coding albumen is considered first, if a gene has multiple transcripts to encode egg It is white, then the most corresponding transcript of albumen of preferred number containing amino acid, if multiple transcript amino acid contents are identical, into one The most transcript of step selection number containing base.Upper principle accordingly, the GUCA1A gene transcripts number that we are filtered out are as follows: ENST00000394237 (note: the number comes from Ensemble database,www.ensembl.org, transcript coding can be inputted Retrieve transcript details and transcript sequence).
2.1.2 the design of hybridization probe:
The design standard of hybridization probe are as follows: (1) probe cover the target area of all candidate genes, i.e., exon region with And exon and introne stitching portion (each 100 bp of exon upstream and downstream)；(2) repetitive sequence is removed: for going out in genome The repeated fragment of existing highly repetitive sequence and the lower frequency for occurring 2-5 times in human genome, we are removed, It avoids capturing other homologous genes, increases false positive, to reduce detection efficiency.Applicant is by the target area of all candidate genes Domain is compared with human genome DNA's sequence, removes 2.5% repetitive sequence altogether；(3) in probe design process, I Specific integration carried out to the exon closed on, adjacent probe integrates standard are as follows: when the integration objective of neighboring exons Region (i.e. the upstream 100bp of the one before exon, which rises to the downstream 100bp of the latter exon, to be stopped) summation is less than 600bp, i.e., will It is integrated into a probe, in the hope of completing the capture of multipair exon region by a pair of of probe；(4) when designed probe sequence When less than 250bp, on the basis of its both ends respectively includes the introne of upstream and downstream 100bp, the interior of identical bp number is respectively continued growing Containing son, probe size is made to reach 250bp.According to the above design principle, we are for probe sequence designed by GUCA1A gene It is as follows:
For screening hybridization probe sequence totally 16 of CACD Disease-causing gene GUCA1A, sequence such as SEQ ID NO.1~SEQ Shown in ID NO.16；
The capture of 2.2 target areas and deep sequencing:
First by genomic DNA fragment, and at DNA end mark " A ", with Illumina PE connector-few nucleosides acid-mixed It closes object to be connected, connection product is enriched with through PCR, obtains DNA library.Then DNA library and known Disease-causing gene are captured into chip Hybridization, elution, purifying, obtain coded sequence.Finally creation pairing end, in HiSeqTM 2000 (Illumina, Inc., San Diego, CA, USA) target sequence is sequenced on platform.
2.3 pairs of sequencing datas carry out bioinformatic analysis, filter out candidate disease causing genes:
2.3.1 using Mosaik software (http://bioinformatics.bc.edu/marthlab/Mosaik) processing Illumina raw sequencing data (pairing end data), generates .bam type file..bam file is inputted into GATK, is utilized GATK detects single nucleotide variations body (single nucleotide variant) and small insertion or missing (insertion/ Deletions), while quality evaluation is carried out, convenient for the bioinformatic analysis in downstream, finally generates .vcf type file.
It 2.3.2 is including dbSNP144 by the sequencing result of patient
(http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/ Snp144.txt.gz.), HapMap plans
(ftp: //ftp.ncbi.nlm.nih.gov/hapmap), 1000Genome Project
(ftp: //ftp.1000genomes.ebi.ac.uk/vol1/ftp), Yan Di and Huang Di, two legendary rulers of remote antiquity's database (http: // ) and Exome yh.genomics.org.cn/
Five mononucleotides including Variant Server (http://evs.gs.washington.edu/EVS/) are more Screening in state property (SNP) database filters all known SNP sites；
2.3.3 gene order corresponding to the sequencing result by patient is compared and analyzes, and preferentially analyzes insertion/deletion Mutation, nonsense mutation and missense mutation, as a result can be divided into three classes, new mutation and new gene including known mutations, known Mutation.
2.4, through Sanger sequence verification, identify Disease-causing gene:
PCR method is directed to the mutational site filtered out respectively and neighbouring DNA sequence dna is expanded in corresponding family, used to draw Object sequence is designed using Primer 3 (http://frodo.wi.mit.edu/) primer-design software.The reactant of PCR used It is (20 μ L system) are as follows: 4 μ L, 25mM MgCl of 5*buffer22 μ L, DNA 1 μ L, forward primer F 1 μ L, 1 μ of reverse primer R L, 10mM dNTP0.4 μ L, Taq enzyme 0.1 μ L, ddH2O 10.5μL.PCR response procedures: 98 DEG C of 5min, 35 recycle (98 DEG C 10s, 60 DEG C of 15s, 72 DEG C of 1min), 72 DEG C of 7min, 4 DEG C of 5min.1% agarose gel electrophoresis detection, under ultraviolet light bale cutting instrument It cuts PCR product gel and purifies.All PCR products are sequenced respectively with forward and reverse primer, and to sequencing result carry out into One step analysis, compared online using NCBI tools BLAST (http://blast.ncbi.nlm.nih.gov/), exclude false positive As a result, and filtering out the mutational site isolated in family.Wherein detect 42146547 and 42146548 nucleotide sites Forward primer sequence is SEQ ID NO.17, and reverse primer sequences are SEQ ID NO.18.
Experimental result:
1. family clinical data:
Clinical ophthalmology expert in the family participation research all patients (II:6, II:11, II:13, III:7, III:12, III:14, III:16, III:18, III:20, III:31, III:33, IV:3, IV:5, IV:10, IV:13 and IV: 19) after having carried out clinical examination comprehensive in detail, the clinical diagnosis of " CACD " has been made to patient in the family.Patient in family The eyeground of CACD each clinical stages shines, inspection result such as Fig. 2-Fig. 5: Fig. 2 of autofluorescence and fluoroscopic visualization is patient IV:10's Examination of eyes is as a result, meet the performance of 1 phase of CACD clinical stages；Fig. 3 is that the examination of eyes of patient III:16 faces as a result, meeting CACD 2 phases showed bed by stages；Fig. 4 is the examination of eyes of patient III:14 as a result, meeting the performance of 3 phase of CACD clinical stages；Fig. 5 is patient The examination of eyes of III:18 is as a result, meet the performance of 4 phase of CACD clinical stages.The Detailed clinical data of all patients is such as in family Under:
The result of clinical detection of patient in 1. family of table
2. the family Genetic Detection result:
By having carried out full sequencing of extron group and bioinformatic analysis to two patients IV:3 and IV:13 in family Afterwards, it has been found that two patients carry suspicious heterozygous mutant GUCA1A p.Arg120Leu, correspond to nucleotide and change Become GUCA1A c.359_369delGCinsTT, does not find other suspicious Disease-causing gene mutational sites.It is sequenced through Sanger Verifying confirms that the mutational site shows as isolating in the family, and sequencing result is shown in Fig. 6.It is mutated GUCA1A p.Arg120Leu Positioned at No. 6 chromosomes, physical location is that the base of 42146547 and 42146548 (Ensemble databases) sports TT by GC； Rna level: GUCA1A gene coding RNA the 359th and 360 bit bases sport TT by GC；Protein level: SPP2 gene encodes egg White 120th amino acids become leucine from arginine, and the mutation of the gene-correlation is never found in RP patient.
Screening process according to designed by us, by the genetic chip and deep sequencing technology designed by us, we Success confirms that the detected GUCA1A genetic heterozygosis sports the new Disease-causing gene of CACD, and p.Arg120Leu is the disease New pathogenic sites.
Embodiment 2:
Function assessment research is carried out for pathogenic mutation GUCA1A p.Arg120Leu detected in embodiment 1.
Experimental method:
1. conservative Analysis:
Using NCBI HomoloGene database (http://www.ncbi.nlm.nih.gov/homologene) to institute Screening obtains being mutated carries out conservative assessment and prediction in multiple species.
2. albumin crystal structure changes research:
Using SWISS MODEL (http://swissmodel.expasy.org/) forecasting software to GUCA1A wild type Albumen and the mutain structure for carrying p.Arg120Leu mutation are predicted that assessment is mutated caused albumen knot respectively Structure changes.
3. zebra fish is tested:
It is respectively synthesized wild type and saltant type source of people GUCA1A full length gene cDNA and is cloned into pxT7 carrier, construct The GUCA1A of pxT7 labelWTAnd GUCA1AMUPlasmid synthesizes corresponding GUCA1A mRNA, including GUCA1A using these plasmidsWT And GUCA1AMUmRNA.The mRNA of above-mentioned synthesis is carried out to microinjection into zebrafish embryo respectively, observed:
1) change of om observation zebra fish configuration structure；
2) change of transmission electron microscope observing zebra fish eye morphosis；
3) change of zebra fish retinal specific gene expression: 4 days after injection zebra fish are collected, detect its view respectively Variation of the transcript of nethike embrane specificity in mRNA and protein level.Trizol method extracts the mRNA of zebra fish, and utilizes reverse It is cDNA that kit, which is recorded, by its reverse transcription, using the expression of QPCR detection retinal specific transcript, is compared GUCA1AWTAnd GUCA1AMUMRNA injection group.4 days zebra fish are fixed, be dehydrated and OCT embedding, to its eyeball tissue Frozen section is carried out, immunofluorescence dyeing is carried out to frozen section, primary antibody is PNA/RHO/ZPR-2 antibody, is added after rinsing Fluorescence secondary antibody and DAPI observe it with Olympus laser confocal microscope after mounting, compare GUCA1AWTWith GUCA1AMUMRNA injection group.
4) variation of zebra fish choroidal artery morphosis: 10 days after injection Tg (flk1:EGFP) transgenosis is collected Zebra fish compares GUCA1A using its choroidal artery of Leica confocal laser scanning microscopeWTAnd GUCA1AMUMRNA note Penetrate group.
Experimental result:
1. conservative Analysis:
The site GUCA1A p.Arg120Leu is highly protected in multiple species such as people, orangutan, wolf, ox, mouse, chicken and zebra fish It keeps, i.e., the site is highly conserved during evolution, and the mutation that the site is confirmed in side may cause more serious pathology Phenomenon (Fig. 7).
2. albumin crystal structure changes research:
Albumin crystal structure prediction is the results show that GUCA1A p.Arg120Leu mutation can cause the site wild type Arg120 Hydrogen bond structure between Ser51 amino acid disappears, and causes the change (Fig. 8) of protein structure, to further influence albumen Function.
3. zebra fish experimental result:
We compare GUCA1A from the following aspectsWTAnd GUCA1AMUThe similarities and differences of mRNA injection group zebra fish:
1) zebra fish configuration structure: GUCA1AWTAnd GUCA1AMUMRNA injection group zebra fish is in configuration structure On there is not significant difference (Fig. 9)；
2) change of zebra fish eye morphosis: we are by means of transmission electron microscope to GUCA1AWTAnd GUCA1AMU mRNA The eye morphosis of injection group zebra fish compares, and finds GUCA1AMUThe RPE of mRNA injection group zebra fish obviously becomes Thin, photoreceptor cell outer segment disc layer morphosis is abnormal (Figure 10)；
3) change of zebra fish retinal specific gene expression: we compare GUCA1AWTAnd GUCA1AMUMRNA note The differential expression of group zebra fish retinal specific gene transcripts and albumen is penetrated, finds GUCA1AMUMRNA injection group zebra fish Retinal specific transcript is expressed compared with GUCA1AWTMRNA injection group significantly reduces.At the same time, we also to injection after Zebra fish has carried out ocular tissue's slice immunofluorescence dyeing, and the rod cell of zebra fish is marked with RHO, marks zebra with PNA The layer of retina,pigment epithelium of zebra fish is marked with ZPR-2 for the cone cell of fish, the results show that injection GUCA1AMU mRNA Zebra fish rod cell layer (RHO label), cone cell layer (PNA label) and layer of retina,pigment epithelium (ZPR-2 mark Note) lack, and each cellular layer of the zebra fish for injecting wild type mRNA is more complete (Figure 11).To sum up, zebra fish experimental result Show GUCA1AMUMRNA can cause the special damage of zebra fish photoreceptor and RPE cell, in this result and patient The result arrived is similar.
4) variation of zebra fish choroidal artery morphosis: we compare GUCA1AWTAnd GUCA1AMUMRNA injection The morphosis of group zebra fish choroidal artery, finds GUCA1AMUMRNA injection group zebra fish choroidal artery morphosis is different Often, the fluorescence intensity of blood vessel is compared with GUCA1AWTMRNA injection group zebra fish significantly reduces (Figure 12).
To sum up, we confirm GUCA1A by the experiment conclusion of science of heredity and zoopery from many aspects The retina of p.Arg120Leu mutation and choroid special damage, illustrate its being associated between CACD disease.Institute of the present invention The GUCA1A gene for the mutation stated or 1 albumen of GCAP of the mutation can be in preparation heredity centrality halo shape views It is applied in film lesion detection reagent or detection device.

Claims (11)

1. a kind of GUCA1A gene of mutation relevant with heredity centrality halo shape retinopathy, it is characterised in that wild Gene number of the type GUCA1A gene in Ensemble database are as follows: ENSG00000048545, the GUCA1A of the mutation Gene sports T by G in the base that physical location is 42146547, is sported in the base that physical location is 42146548 by C T, other parts are identical as wild type.
2. a kind of 1 albumen of GCAP of mutation relevant with heredity centrality halo shape retinopathy, 1 egg of wild type GCAP The white gene transcripts number in Ensemble database are as follows: ENST00000394237,1 albumen of GCAP of mutation is in the open country 120th amino acids of raw type albumen become leucine from arginine, and other parts are identical as wild type.
3. detecting the GUCA1A gene of mutation described in claim 1 or 1 albumen of GCAP of mutation as claimed in claim 2 Reagent preparation heredity centrality halo shape retinopathy detection reagent or detection device in application.
4. application according to claim 3, it is characterised in that the detection reagent is selected from: primer or primer pair, probe, One of antibody or nucleic acid chip are a variety of.
5. application according to claim 3, it is characterised in that the detection device includes containing detection mutation The detection platform of the genetic chip of GUCA1A gene.
6. a kind of kit for detecting heredity centrality halo shape retinopathy, it is characterised in that the kit packet It includes:
(a) detection GUCA1A gene physical location is the reagent of 42146547 and 42146548 nucleotide sites；Or detection GCAP The reagent in the 120th amino acids site of 1 albumen；
(b) products instruction.
7. kit according to claim 6, it is characterised in that the reagent is selected from primer or primer pair, probe, resists One of body or nucleic acid chip are a variety of.
8. kit according to claim 7, it is characterised in that the reagent is the base for being platform based on deep sequencing Because of chip hybridization probe.
9. kit according to claim 8, it is characterised in that 42146547 and 42146548 nucleotide sites of detection Hybridization probe sequence is chr6 | 42146509-42146628, sequence is as shown in SEQ ID NO.14.
10. kit according to claim 6, it is characterised in that the reagent is detection 42146547 and 42146548 The primer pair of nucleotide site.
11. kit according to claim 6, it is characterised in that 42146547 and 42146548 nucleotide sites of detection Forward primer sequence is SEQ ID NO.17, and reverse primer sequences are SEQ ID NO.18.
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CN108034709B (en) * 2017-12-18 2021-05-07 青岛大学 Application of GUCA1A gene in preparation of products for detecting cone cell malnutrition
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Disease progression in autosomal dominant cone–rod dystrophy caused by a novel mutation (D100G) in the GUCA1A gene;Eva Nong，et al.;《Doc Ophthalmol》;20131219;第128卷;第59-67页 *
Novel GUCA1A mutation identified in a Chinese family with cone-rod dystrophy;Li Huang,et al.;《Neuroscience Letters》;20131231;第541卷;第179-183页 *

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