CN103667438A - Method for screening HRDs disease-causing mutation and gene chip hybridization probe designing method involved in same - Google Patents
Method for screening HRDs disease-causing mutation and gene chip hybridization probe designing method involved in same Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines, and relates to a method for screening HRDs disease-causing mutation and a gene chip hybridization probe designing method involved in the same. The method for screening the HRDs disease-causing mutation comprises the steps of (1) establishing an HRDs genetic resource repository; (2) designing and synthesizing a gene chip hybridization probe of an HRDs disease-causing gene, and integrating the gene chip hybridization probe onto a gene chip; (3) capturing a target area by utilizing the prepared gene chip and executing the depth sequencing; (4) analyzing the sequencing data on the aspect of bioinformatics, and screening the candidate disease-causing gene; (5) functionally predicting a newly-discovered splicing gene mutation site. By establishing the high-efficient HRDs target gene capturing technology, adopting the depth sequencing as a means and confirming the efficiency of the HRDs capturing chip, a high-efficient credible biological information analysis model is established.
Description
Technical field
The invention belongs to biomedicine field, a kind of gene chip hybridization probe design method that relates to method of examination HRDs pathogenic mutation and relate to.
Background technology
Retinal hereditary disease (Hereditary retinal diseases, HRDs) is one group of carrying out property retina degenerative disease being caused by hereditary defect, is common and endanger serious heredity blinding disease clinically.As the first diseases causing blindness of the age groups of working in world wide, HRDs is about 1/3000 at American-European sickness rate, in China especially up to 1/1000.China is HRDs genetic resources big country, but the genetics information that HRDs is relevant is at present many from western countries, and therefore China HRDs patient is carried out to deep genetics research seems particularly important.
HRDs mostly is single gene inheritance disease, and numerous genetic flaws all can cause it to occur, and its common hereditary pattern has autosomal dominant inheritance, autosomal recessive inheritance and x linked recessive heredity.So far, the whole world has identified 191 HRDs related genes and 231 chain sites (www.RetNet.org), and along with research is goed deep into, the positive cumulative year after year of this number.More than its Disease-causing gene number, show the patient that clinical manifestation is identical, probably have different genotype, be i.e. significant genetic heterogeneity.Still have at present the Disease-causing gene that is greater than 60% (western countries' statistics, China's ratio is higher) HRDs patient not yet to find, prompting exists a large amount of new Disease-causing genes to have to be excavated.
By DNA, transcribing the precursor RNA (pre-mRNA) obtaining needs to become further participation translation process synthetic protein of messenger RNA(mRNA) (mRNA) through montage, and this montage process mainly occurs in spliceosome.The super large molecular complex that spliceosome is comprised of small molecular core ribonucleoprotein (snRNPs), the snRNP that forms spliceosome has five kinds: U1, U2, U4/U6 and U5.Research points out, the gene (shearing gene) relevant to precursor RNA spliceosome encoding histone can cause autosomal dominant retinitis pigmentosa (ADRP).Be proved at present that the shearing gene that can cause ADRP has PRPF3, PRPF6, PRPF8, PRPF31, RP9 and by the SNRNP200 that applicant found and reported, these shearing genes all cause ADRP by affecting U4/U6-U5 complex body.It is worth noting, above-mentioned six albumen wide expression in the various cells of organism of shearing coded by said gene, but research at present only finds that the sudden change of these six gene-correlations can cause retinal diseases, does not cause the report of other diseases.Therefore, for the correlative study of shearing gene and RP morbidity, seem particularly necessary.
Research for HRDs must be based upon on the basis of certain Protocols in Molecular Biology.A free-revving engine of research HRDs Disease-causing gene is to carry out HRDs molecular diagnosis, and in view of its significant genetic heterogeneity, how detecting numerous Disease-causing gene sudden changes is one of current difficult problems.Positional cloning strategy based on linkage analysis is the classical way of identifying single gene inheritance disease Disease-causing gene, but also faces some difficulties simultaneously: 1. conventionally need to be many for family, be difficult to analyze little family and Sporadic cases.2. sometimes how for family, can not locate pathogenic sites.3. be difficult to filter out correct Disease-causing gene in desmic region.Therefore,, in view of the character of HRDs disease itself and the limitation of traditional analysis technology, the research method of seeking a kind of brand-new HRDs Disease-causing gene seems particularly urgent.
Summary of the invention
The object of the invention is for above-mentioned defect, a kind of method of design of degree of depth order-checking as the hybridization probe of the related gene chip of platform examination HRDs pathogenic mutation of take is provided.
Another object of the present invention is to provide a kind of method of degree of depth order-checking as platform examination HRDs pathogenic mutation of take.
Another object of the present invention is to provide new HRDs Disease-causing gene.
Object of the present invention can be achieved through the following technical solutions:
Take degree of depth order-checking as platform examination or detect the method for design of the related gene chip hybridization probe of HRDs pathogenic mutation, comprising:
(1) selection of candidate gene: described gene trap chip has been contained whole 179 retinal diseases genes involveds of being announced by RetNet, and the shearing gene that highly suspection may be relevant to retinal diseases;
(2) selection of transcript: select particular transcripts for different genes, selection principle is: the transcript of first considering to have CCDS proteins encoded, if a gene has the equal proteins encoded of a plurality of transcripts, first-selected containing the maximum corresponding transcript of albumen of amino acid no, if a plurality of transcript aminoacids contents are identical, further select containing the maximum transcript of base number;
(3) design of hybridization probe: according to the different transcript design hybridization probes of picking out in (2), standard is: (a) probe covers exon region and exon and the intron stitching portion of all candidate genes; (b) remove the highly repetitive sequence occur in human genome and occur the repeated fragment of 2-5 lower frequency doubly; (c) to closing on the probe of exon, integrate, integration standard is: when the integration objective region of adjacent exon summation is less than 600bp, is about to it and is integrated into a probe, in the hope of completing catching of multipair exon region by a pair of probe; Wherein, the integration objective region of described adjacent exon refer to the upstream 100bp of front exon rise to the downstream 100bp of a rear exon only; (d) when designed probe sequence is less than 250bp, at its two ends, respectively comprise on the basis of intron of upstream and downstream 100bp, the intron that each continues to increase identical bp number, makes probe size reach 250bp.
Wherein, described height is suspected the preferred SNRNP40 of shearing gene, SNRNP27, PRPF4 and EFTUD2 that may be relevant to retinal diseases.The inventive method is a kind of general hybridization probe method of design of degree of depth order-checking as the related gene chip of platform examination HRDs pathogenic mutation of take, described height suspects that shearing gene that may be relevant to retinal diseases is not limited to above-mentioned preferred 4 genes, can be also other height suspect may be relevant to retinal diseases shearing gene.
Described exon and intron stitching portion refer at least each 100 bp of exon upstream and downstream.
In the gene chip hybridization probe design method that to the present invention is based on degree of depth order-checking be Platform Screening HRDs Disease-causing gene, design for screening totally 14 of the hybridization probe sequences of HRDs Disease-causing gene SNRNP40, sequence is as shown in SEQ ID NO.1 ~ SEQ ID NO.14; For screening totally 7 of the hybridization probe sequences of HRDs Disease-causing gene SNRNP27, sequence is as shown in SEQ ID NO.15 ~ SEQ ID NO.21; For screening totally 15 of the hybridization probe sequences of HRDs Disease-causing gene PRPF4, sequence is as shown in SEQ ID NO.22 ~ SEQ ID NO.36; For screening totally 28 of the hybridization probe sequences of HRDs Disease-causing gene EFTUD2, sequence is as shown in SEQ ID NO.37 ~ SEQ ID NO.64; For screening totally 16 of the hybridization probe sequences of HRDs Disease-causing gene PRPF3, sequence is as shown in SEQ ID NO.65 ~ SEQ ID NO.80; For screening totally 43 of the hybridization probe sequences of HRDs Disease-causing gene SNRNP200, sequence is as shown in SEQ ID NO.81 ~ SEQ ID NO.123; For screening totally 8 of the hybridization probe sequences of HRDs Disease-causing gene RP9, sequence is as shown in SEQ ID NO.124 ~ SEQ ID NO.131; For screening totally 44 of the hybridization probe sequences of HRDs Disease-causing gene PRPF8, sequence is as shown in SEQ ID NO.132 ~ SEQ ID NO.175; For screening totally 12 of the hybridization probe sequences of HRDs Disease-causing gene PRPF31, sequence is as shown in SEQ IDNO.176 ~ SEQ IDNO.187; For screening totally 21 of the hybridization probe sequences of HRDs Disease-causing gene PRPF6, sequence is as shown in SEQ ID NO.188 ~ SEQ ID NO.208.
The method that the degree of depth order-checking of take is platform examination HRDs pathogenic mutation, comprises the following steps:
(1) set up HRDs genetic resource, collect HRDs class patient's clinical data and blood preparation, extract genomic dna;
(2) according to above-mentioned hybridization probe method of design, design and synthesize the hybridization probe of HRDs Disease-causing gene chip, and be integrated on gene chip;
(3) utilize the gene chip target acquisition region of preparation and carry out degree of depth order-checking;
(4) bioinformatic analysis sequencing result being optimized, filters out highly suspicious Disease-causing gene and pathogenic mutation;
(5) for newfound shearing gene mutation site, carry out pathogenecity prediction and functional study.
The gene chip that the described preferred Roche Nimblegen of the gene chip company of step (2) produces.
Gene chip target acquisition region prepared by the described utilization of step (3) is also carried out degree of depth order-checking and is preferably utilized the Hi-seq2000 instrument of American I llumina company to complete.
Gene chip target acquisition region prepared by the described utilization of step (3) is also carried out degree of depth order-checking preferred flow and is: by genomic DNA fragment, at DNA end mark " A " and with Illumina PE joint-oligonucleotide mixture, be connected; Connect product through PCR enrichment, obtain DNA library, and chip hybridization, wash-out, purifying are caught in DNA library and known Disease-causing gene, obtain encoding sequence; Create pairing end, on IlluminaHiSeqTM2000 platform, target sequence is checked order.
Step (4) is described carries out bioinformatic analysis to sequencing data and preferably includes:
(1) adopt Mosaik software processes Illumina primitive sequencer data, produce .bam type file, by .bam file input GATK, utilize GATK to detect single nucleotide variations body and little insertion or disappearance, carry out quality evaluation simultaneously, be convenient to the bioinformatic analysis in downstream, finally produce .vcf type file;
(2) patient's sequencing result is being comprised to dbSNP132
(http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/snp132. txt.gz.), HapMap plan
(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://yh.genomics.org.cn/) and Exome Variant Server (http://evs.gs.washington.edu/EVS/) examination in interior five single nucleotide polymorphism (SNP) database, filters all known SNP sites;
(3) the corresponding gene order of patient's sequencing result is compared and analyzed, the sudden change of precedence parse insertion/deletion, nonsense mutation and missense mutation, result can be divided three classes, and comprises the sudden change of new mutant and the new gene of known mutations, known.
The HRDs Disease-causing gene obtaining by method of the present invention is the shearing gene relevant to retinal diseases, is respectively SNRNP40, SNRNP27, PRPF4 and EFTUD2.
Beneficial effect
1.HRDs is common, serious heredity blinding disease, at China's sickness rate, up to 1/1000, is the first diseases causing blindness in work age groups in world wide.Excavate the new pathogenic mutation of HRDs and the Molecular etiology that new Disease-causing gene is conducive to further explore HRDs, it is the ophthalmology inherited disease resource that makes full use of China, benefiting retinal hereditary disease patient's reality needs, is one of most important research direction of genome times afterwards comprehensively.This patent is intended to explore the genetics cause of disease of HRDs, thereby helps to understand pathogenesis, auxiliary clinical diagnosis, antenatal diagnosis and therapeutic transgene.
2. studies confirm that in a large number, the dysfunction of shearing gene can cause ADRP.Therefore, for the correlative study of shearing gene and ADRP, seem particularly necessary.The candidate gene of this patent has been contained 6 known pathogenic shearing genes and 4 highly suspicious shearing genes, all genes are all by applicant, on the basis of being engaged in for many years genetics research, to consult lot of documents to filter out, by the relation to further clear and definite these genes and HRDs of the inventive method.
3. gene can corresponding a plurality of different transcripts, and coded RNA and the albumen of different transcript is different.In this patent, applicant, according to the experience of being engaged in for a long time genetics research, filters out optimum transcript, and designs corresponding probe according to different transcripts, thereby makes examination benefit reach the highest.
4. the exon region of gene changes, and can directly cause that aminoacid sequence changes, thereby cause the structure and function of albumen to change.Yet study, show, the change of exon and intron junction region base can directly affect the shearing of gene.The designed hybridization probe of this patent covers exon region and exon and intron stitching portion (exon upstream and downstream is each 100 bp at least) of all known Disease-causing genes, makes pathogenic sites examination face reach the widest.
5. in probe design process, this patent is integrated the exon closing on, be intended to a probe in detecting 600bp with the integration objective region of interior adjacent exon (the upstream 100bp of front exon rise to the downstream 100bp of a rear exon only), in the hope of completing catching of two pairs of even multipair exon regions by a pair of probe, make the screening efficiency of probe reach the highest.Meanwhile, the repeated fragment that we have rejected highly repetitive sequence contained in target area and occur 2-5 lower frequency doubly in human genome, avoids catching other homologous genes, makes the false positive rate of examination drop to minimum.
6.HRDs has significant genetic heterogeneity, 191 of current known Disease-causing genes, and 231, chain site, and still there is the Disease-causing gene of a large amount of the unknowns.The research of application conventional art obviously has significant limitation.Degree of depth sequencing technologies is a kind of high throughput sequencing technologies of comparative maturity, can disposablely to magnanimity gene (full genome), carry out parallel order-checking, has the incomparable advantage of conventional art.Studies confirm that degree of depth sequencing technologies has huge advantage aspect the gene excavating of Mendelian inheritance disease and Disease-causing gene examination, by degree of depth sequencing technologies be applied to China's retinal hereditary disease research be very important, feasible.The patent that we apply for is intended to set up efficient HRDs target gene capture technique, take degree of depth order-checking as means, confirms that HRDs catches chip efficiency, sets up efficient, believable analysis of biological information model.
Accompanying drawing explanation
Retinal diseases genes involved and number of loci that Figure 119 80-2012 has identified
Fig. 2 excavates the technological line of new Disease-causing gene
Fig. 3 degree of depth order-checking flow process
Fig. 4 pedigree chart (red frame is labeled as the patient that takes a blood sample)
Fig. 5 propositus eyeground is shone
Fig. 6 sequencer map
The prediction of Fig. 7 protein structure
Embodiment
Embodiment 1
Experimental technique:
The foundation of 1.HRDs genetic resource.
1.1 collect following three class patients' clinical data and blood preparation:
1.1.13 generation or autosomal dominant inheritance familys more than 3 generations, autosomal recessive inheritance family, x linked recessive Genetic Families, comprise RP, a Leber Xian Xing ?Mongolian, congenital stationary night blindness, yolk sample macular dystrophy, Stargardt sick.
1.1.2 collect the little family of heredity of various HRDs.
1.1.3 collect the Sporadic cases without the various HRDs of family history.
1.2 extraction genomic dna:
Adopt TIANamp blood DNA extraction agent box (Tiangen Biotech Co.Ltd, Beijing, China), the protocol providing according to producer extracts patient's genomic dna from the patient's peripheral blood collecting.
2. excavate the new mutant (seeing Fig. 2) of HRDs new Disease-causing gene/known Disease-causing gene.
2.1 design and customize HRDs genes involved catches chip:
2.1.1 the selection of candidate gene:
This gene trap chip has been contained whole 179 retinal diseases genes involveds of being announced by RetNet (comprising 6 known pathogenic shearing genes), and 4 shearing genes that highly suspection may be relevant to retinal diseases.Wherein, 6 known pathogenic shearing genes are respectively: PRPF3, PRPF6, PRPF8, PRPF31, RP9 and SNRNP200 (this gene by applicant take the lead in discovery report and HRDs disease-related); 4 highly suspicious shearing genes are respectively: SNRNP40, SNRNP27, PRPF4 and EFTUD2, its gene numbering in Ensemble database is as shown in the table.Above gene is all by applicant, on the basis of being engaged in for many years genetics research, to consult lot of documents to filter out.
| Gene | Gene numbering | Gene | Gene numbering |
| PRPF3 | ENSG00000117360 | SNRNP200 | ENSG00000144028 |
| PRPF6 | ENSG00000101161 | SNRNP40 | ENSG00000060688 |
| PRPF8 | ENSG00000174231 | SNRNP27 | ENSG00000124380 |
| PRPF31 | ENSG00000105618 | PRPF4 | ENSG00000136875 |
| RP9 | ENSG00000164610 | EFTUD2 | ENSG00000108883 |
Note: above information all from Ensemble database (
www.ensembl.org), can input genes encoding retrieval gene details and gene order.
2.1.2 the selection of transcript:
For different genes, select specific transcript, each gene all contains a plurality of transcripts, when selecting transcript, our principle is: the transcript of first considering to have CCDS proteins encoded, if a gene has the equal proteins encoded of a plurality of transcripts, first-selected containing the maximum corresponding transcript of albumen of amino acid no, if a plurality of transcript aminoacids content is identical, further select containing the maximum transcript of base number.Go up according to this principle,
10 shearing gene-correlation transcripts that we filter out are respectively:
| Gene | Transcript numbering | Gene | Transcript numbering |
| PRPF3 | ENST00000324862 | SNRNP200 | ENST00000323853 |
| PRPF6 | ENST00000266079 | SNRNP40 | ENST00000263694 |
| PRPF8 | ENST00000572621 | SNRNP27 | ENST00000244227 |
| PRPF31 | ENST00000321030 | PRPF4 | ENST00000374198 |
| RP9 | ENST00000297157 | EFTUD2 | ENST00000426333 |
Note: above information all from Ensemble database (
www.ensembl.org), can input transcript coding retrieval transcript details.
2.1.3 the design of hybridization probe:
Applicant is according to the different transcript design hybridization probes of picking out, the customization of Bing You Roche-NimbleGen company.The standard of hybridization probe is: (1) probe covers the target area of all candidate genes, i.e. exon region and exon and intron stitching portion (each 100 bp of exon upstream and downstream); (2) remove tumor-necrosis factor glycoproteins: for the highly repetitive sequence occurring at genome and the repeated fragment that occurs 2-5 lower frequency doubly in human genome, we are removed, avoid catching other homologous genes, increase false positive, thereby reduce detection efficiency.Applicant compares the target area of all candidate genes and human genome DNA's sequence, has removed altogether 2.5% tumor-necrosis factor glycoproteins; (3) in probe design process, we have carried out specific integration to the exon closing on, its adjacent probe integration standard is: when the integration objective region of adjacent exon (the upstream 100bp of an exon rise to after an exon downstream 100bp only) summation is less than 600bp, be about to it and be integrated into a probe, in the hope of completing catching of multipair exon region by a pair of probe; (4) when designed probe sequence is less than 250bp, at its two ends, respectively comprise on the basis of intron of upstream and downstream 100bp, the intron that each continues to increase identical bp number, makes probe size reach 250bp.According to above principle of design, it is as follows that we shear the designed probe sequence of gene for 16:
For screening totally 14 of the hybridization probe sequences of HRDs Disease-causing gene SNRNP40, sequence is as shown in SEQ ID NO.1 ~ SEQ ID NO.14; For screening totally 7 of the hybridization probe sequences of HRDs Disease-causing gene SNRNP27, sequence is as shown in SEQ ID NO.15 ~ SEQ ID NO.21; For screening totally 15 of the hybridization probe sequences of HRDs Disease-causing gene PRPF4, sequence is as shown in SEQ ID NO.22 ~ SEQ ID NO.36; For screening totally 28 of the hybridization probe sequences of HRDs Disease-causing gene EFTUD2, sequence is as shown in SEQ ID NO.37 ~ SEQ ID NO.64; For screening totally 16 of the hybridization probe sequences of HRDs Disease-causing gene PRPF3, sequence is as shown in SEQ ID NO.65 ~ SEQ ID NO.80; For screening totally 43 of the hybridization probe sequences of HRDs Disease-causing gene SNRNP200, sequence is as shown in SEQ ID NO.81 ~ SEQ ID NO.123; For screening totally 8 of the hybridization probe sequences of HRDs Disease-causing gene RP9, sequence is as shown in SEQ ID NO.124 ~ SEQ ID NO.131; For screening totally 44 of the hybridization probe sequences of HRDs Disease-causing gene PRPF8, sequence is as shown in SEQ ID NO.132 ~ SEQ ID NO.175; For screening totally 12 of the hybridization probe sequences of HRDs Disease-causing gene PRPF31, sequence is as shown in SEQ ID NO.176 ~ SEQ ID NO.187; For screening totally 21 of the hybridization probe sequences of HRDs Disease-causing gene PRPF6, sequence is as shown in SEQ ID NO.188 ~ SEQ ID NO.208.
Catch and degree of depth order-checking (seeing Fig. 3) 2.2 target areas:
First by genomic DNA fragment, and at DNA end mark " A ", be connected with Illumina PE joint-oligonucleotide mixture, connect product through PCR enrichment, obtain DNA library.Then chip hybridization, wash-out, purifying are caught in DNA library and known Disease-causing gene, obtain encoding sequence.Finally create pairing end, on Illumina HiSeqTM2000 platform, target sequence is checked order.
2.3 pairs of sequencing datas carry out bioinformatic analysis, filter out candidate's Disease-causing gene:
2.3.1 adopt Mosaik software (
http:// bioinformatics.bc.edu/marthlab/Mosaik) process Illumina primitive sequencer data (pairing end data), produce .bam type file.By .bam file input GATK, utilize GATK to detect single nucleotide variations body (single nucleotide variant) and little insertion or disappearance (insertion/deletions), carry out quality evaluation simultaneously, be convenient to the bioinformatic analysis in downstream, finally produce .vcf type file.
2.3.2 patient's sequencing result is being comprised to dbSNP132
(http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/snp132. txt.gz.), HapMap plan
(
ftp: //fp.ncbi.nlm.nih.gov/hapmap), 1000Genome Project (
ftp: //ftp.1000genomes.ebi.ac.uk/vol1/fp), Yan Di and Huang Di, two legendary rulers of remote antiquity's database (
http:// yh.genomics.org.cn/) and Exome Variant Server (
http:// evs.gs.washington.edu/EVS/) examination in interior five single nucleotide polymorphism (SNP) database, filter all known SNP sites;
2.3.3 the corresponding gene order of patient's sequencing result is compared and analyzed, the sudden change of precedence parse insertion/deletion, nonsense mutation and missense mutation, result can be divided three classes, and comprises the sudden change of new mutant and the new gene of known mutations, known.
2.4 through Sanger sequence verification, identifies Disease-causing gene:
PCR method increases in corresponding family for the mutational site filtering out and contiguous DNA sequence dna respectively, and the primer sequence adopts the design of Primer3 (http://frodo.wi.mit.edu/) primer-design software.The reaction system of PCR used (20 μ L system) is: 5*buffer4 μ L, 25mMMgCl
22 μ L, DNA1 μ L, F (upstream primer) 1 μ L, R (downstream primer) 1 μ L, 10mM dNTP0.4 μ L, Taq enzyme 0.1 μ L, ddH
2o10.5 μ L.PCR response procedures: 98 ℃ of 5min, 35 circulations (98 ℃ of 10s, 60 ℃ of 15s, 72 ℃ of 1min), 72 ℃ of 7min, 4 ℃ of 5min.3% agarose gel electrophoresis detects, and under ultraviolet bale cutting instrument, cuts PCR product gel purifying.To all PCR products, with forward and reverse primer, send the order-checking of Shenzhen Hua Da genome company respectively.And sequencing result is further analyzed, use NCBI to contrast online tools BLAST
(
http:// blast.ncbi.nlm.nih.gov/), get rid of false positive results, and filter out in family, be divided into from mutational site.
Experimental result:
Applicant to 60 patients that made a definite diagnosis of the HRDs family from 45 different hereditary forms of having collected and the normal people in family (for it is numbered HD01-HD40, HD51-HD70) method checking order by above-mentioned target area detects its Disease-causing gene, obtains following initial experiment result:
Note: above mutational site is the physical location of mutating alkali yl correspondence in Ensemble database; In mutation type, Het represents heterozygous mutant, and Hom represents homozygous mutation.
Sudden change with PRPF4 in above table in HD14 patient's family is verified as example, elaborates sudden change checking related experiment result:
1. clinical data
1.1 pedigree charts (seeing Fig. 4)
Propositus's clinical data in 1.2 families:
UCVA: right eye index 50cm, left eye index 30cm, eyes are corrected defects of vision without improving; Eye-ground photography: see Fig. 5; Eyes ERG: eyes a, b ripple all disappear, and are blanking wave.
2. family the result
We extract genomic dna from propositus and relevant kinsfolk's blood, by the patient to all in family, carry out target area order-checking, and we have found our PRPF4p.P315L, and the sudden change of this gene-correlation is never found in HRDs patient.Through Sanger sequence verification confirm to show as in the Gai family of this mutational site be divided into from, and corresponding sudden change is not found in our screening in 200 normal peoples (400 allelotrope).Sequencing result is shown in Fig. 6.
The screening process designed according to us, by us designed gene chip and degree of depth sequencing technologies, we successfully confirm that this mutational site PRPF4p.P315L is new RP pathogenic sites.
Embodiment 2:
For Disease-causing gene detected in embodiment 1, carry out function assessment research, the above-mentioned PRPF4 gene new mutant p.P315L of herein take is example.
Experimental technique:
1. conservative Analysis:
Employing NCBI HomoloGene database (
http:// www.ncbi.nlm.nih.gov/homologene) to screen and obtain sudden change and in a plurality of species, carry out conservative property prediction.
2. according to the pathogenecity of SIFT and the sudden change of PolyPhen value prediction:
Adopt two main flow on-line prediction software: PolyPhen-2 (Polymorphism Phenotyping, version2;
http:// genetics.bwh.harvard.edu/pph2/) and SIFT Human Protein DB (
http:// sift.bii.a-star.edu.sg/), prediction missense mutation and the impact of nonsense mutation on protein level, thereby the pathogenecity of prediction sudden change.
3. albumin crystal structural modification research:
Because PRPF4 gene participate in to form U4/U6 complex body, and this mutational site is arranged in WD40 structural domain, therefore adopt SWISS
MODEL (
http:// swissmodel.expasy.org/)forecasting software carries out structure prediction to PRPF4p.P315L sudden change target spot.
Experimental result:
1. conservative Analysis:
This site of PRPF4p.P315 is equal high conservative in a plurality of species such as yeast, nematode, fish, chicken, mouse, wolf, ox, orangutan and the mankind, i.e. this site high conservative during evolution, thus further prove that the sudden change in this site may cause comparatively serious pathological phenomenon.
2.SIFT and PolyPhen value prediction:
Its SIFT value of PRPF4p.P315L is that 0.07, PolyPhen value is 0.999.Highly point out it to have larger pathogenic possibility.
3. albumin crystal structural modification research:
Studies have shown that this sudden change can cause that obvious protein structure changes, thereby to protein function exert an influence (Fig. 7).
Claims (10)
1. take degree of depth order-checking as the related gene chip hybridization probe design method of platform examination HRDs pathogenic mutation, it is characterized in that comprising:
(1) selection of candidate gene: described gene trap chip has been contained whole 179 retinal diseases genes involveds of being announced by RetNet, and the shearing gene that highly suspection may be relevant to retinal diseases;
(2) selection of transcript: select particular transcripts for different genes, selection principle is: the transcript of first considering to have CCDS proteins encoded, if a gene has the equal proteins encoded of a plurality of transcripts, first-selected containing the maximum corresponding transcript of albumen of amino acid no, if a plurality of transcript aminoacids contents are identical, further select containing the maximum transcript of base number;
(3) design of hybridization probe: according to the different transcript design hybridization probes of picking out in (2), standard is: (a) probe covers exon region and exon and the intron stitching portion of all candidate genes; (b) remove the highly repetitive sequence occur in human genome and occur the repeated fragment of 2-5 lower frequency doubly; (c) to closing on the probe of exon, integrate, integration standard is: when the integration objective region of adjacent exon summation is less than 600bp, is about to it and is integrated into a probe, in the hope of completing catching of multipair exon region by a pair of probe; Wherein, the integration objective region of described adjacent exon refer to the upstream 100bp of front exon rise to the downstream 100bp of a rear exon only; (d) when designed probe sequence is less than 250bp, at its two ends, respectively comprise on the basis of intron of upstream and downstream 100bp, the intron that each continues to increase identical bp number, makes probe size reach 250bp.
2. according to claim 1ly take degree of depth order-checking as the related gene chip hybridization probe design method of platform examination HRDs pathogenic mutation, it is characterized in that the preferred SNRNP40 of shearing gene, SNRNP27, PRPF4 and EFTUD2 that described height suspection may be relevant to retinal diseases, but be not limited to above-mentioned preferred 4 genes, can be also other height suspect may be relevant to retinal diseases shearing gene.
3. according to claim 1ly take degree of depth order-checking as the related gene chip hybridization probe design method of platform examination HRDs pathogenic mutation, it is characterized in that described exon and intron stitching portion refer at least each 100 bp of exon upstream and downstream.
4. according to claim 1ly take degree of depth order-checking as the related gene chip hybridization probe design method of platform examination HRDs pathogenic mutation, it is characterized in that sequence is as shown in SEQ ID NO.1 ~ SEQ ID NO.14 for screening totally 14 of the hybridization probe sequences of HRDs Disease-causing gene SNRNP40; For screening totally 7 of the hybridization probe sequences of HRDs Disease-causing gene SNRNP27, sequence is as shown in SEQ ID NO.15 ~ SEQ ID NO.21; For screening totally 15 of the hybridization probe sequences of HRDs Disease-causing gene PRPF4, sequence is as shown in SEQ ID NO.22 ~ SEQ ID NO.36; For screening totally 28 of the hybridization probe sequences of HRDs Disease-causing gene EFTUD2, sequence is as shown in SEQ ID NO.37 ~ SEQ ID NO.64; For screening totally 16 of the hybridization probe sequences of HRDs Disease-causing gene PRPF3, sequence is as shown in SEQ ID NO.65 ~ SEQ ID NO.80; For screening totally 43 of the hybridization probe sequences of HRDs Disease-causing gene SNRNP200, sequence is as shown in SEQ ID NO.81 ~ SEQ ID NO.123; For screening totally 8 of the hybridization probe sequences of HRDs Disease-causing gene RP9, sequence is as shown in SEQ ID NO.124 ~ SEQ ID NO.131; For screening totally 44 of the hybridization probe sequences of HRDs Disease-causing gene PRPF8, sequence is as shown in SEQ ID NO.132 ~ SEQ ID NO.175; For screening totally 12 of the hybridization probe sequences of HRDs Disease-causing gene PRPF31, sequence is as shown in SEQ ID NO.176 ~ SEQ ID NO.187; For screening totally 21 of the hybridization probe sequences of HRDs Disease-causing gene PRPF6, sequence is as shown in SEQ ID NO.188 ~ SEQ ID NO.208.
5. the method that the degree of depth order-checking of take is platform examination HRDs pathogenic mutation, is characterized in that comprising the following steps:
(1) set up HRDs genetic resource, collect HRDs class patient's clinical data and blood preparation, extract genomic dna;
(2) according to above-mentioned hybridization probe method of design, design and synthesize the hybridization probe of HRDs Disease-causing gene chip, and be integrated on gene chip;
(3) utilize the gene chip target acquisition region of preparation and carry out degree of depth order-checking;
(4) bioinformatic analysis sequencing result being optimized, filters out highly suspicious Disease-causing gene and pathogenic mutation;
(5) for newfound shearing gene mutation site, carry out pathogenecity prediction and functional study.
6. according to claim 5ly take the method that degree of depth order-checking is platform examination HRDs pathogenic mutation, it is characterized in that the described gene chip of step (2) is the gene chip that Roche Nimblegen company produces.
7. according to claim 5ly take the method that degree of depth order-checking is platform examination HRDs pathogenic mutation, it is characterized in that gene chip target acquisition region prepared by the described utilization of step (3) and carrying out degree of depth order-checking is to utilize the Hi-seq2000 instrument of American I llumina company to complete.
8. according to claim 7ly take the method for degree of depth order-checking as platform examination HRDs pathogenic mutation, it is characterized in that utilizing the gene chip target acquisition region of preparation and carry out degree of depth order-checking preferred flow and be: by genomic DNA fragment, at DNA end mark " A " and with Illumina PE joint-oligonucleotide mixture, be connected; Connect product through PCR enrichment, obtain DNA library, and chip hybridization, wash-out, purifying are caught in DNA library and known Disease-causing gene, obtain encoding sequence; Create pairing end, on Illumina HiSeqTM2000 platform, target sequence is checked order.
9. according to claim 5ly take the method that degree of depth order-checking is platform examination HRDs pathogenic mutation, it is characterized in that described sequencing data is carried out to bioinformatic analysis the comprising of step (4):
(1) adopt Mosaik software processes Illumina primitive sequencer data, produce .bam type file, by .bam file input GATK, utilize GATK to detect single nucleotide variations body and little insertion or disappearance, carry out quality evaluation simultaneously, be convenient to the bioinformatic analysis in downstream, finally produce .vcf type file;
(2) patient's sequencing result is being comprised to dbSNP132
(http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/snp132. txt.gz.), HapMap plan
(
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:// yh.genomics.org.cn/) and Exome Variant Server (
http:// evs.gs.washington.edu/EVS/) examination in interior five single nucleotide polymorphism (SNP) database, filter all known SNP sites;
(3) the corresponding gene order of patient's sequencing result is compared and analyzed, the sudden change of precedence parse insertion/deletion, nonsense mutation and missense mutation, result can be divided three classes, and comprises the sudden change of new mutant and the new gene of known mutations, known.
10. the HRDs Disease-causing gene obtaining by method examination claimed in claim 5, is characterized in that described HRDs Disease-causing gene is the shearing gene relevant to retinal diseases, is respectively SNRNP40, SNRNP27, PRPF4, EFTUD2.
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Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105297145A (en) * | 2015-11-06 | 2016-02-03 | 艾吉泰康生物科技(北京)有限公司 | Inherited metabolic disease screening method and reagent kit |
| WO2017106364A3 (en) * | 2015-12-14 | 2017-07-13 | Cold Spring Harbor Laboratory | Compositions and methods for treatment of retinitis pigmentosa 18 and retinitis pigmentosa 13 |
| US9714422B2 (en) | 2014-06-16 | 2017-07-25 | University Of Southampton | Reducing intron retention |
| US9976143B2 (en) | 2014-10-03 | 2018-05-22 | Cold Spring Harbor Laboratory | Targeted augmentation of nuclear gene output |
| CN108342467A (en) * | 2017-01-23 | 2018-07-31 | 黄秀峰 | A kind of diagnostic method, gene probe and its application of MAC Disease-causing genes |
| CN108676865A (en) * | 2018-04-08 | 2018-10-19 | 复旦大学附属眼耳鼻喉科医院 | A kind of glaucoma of childhood related gene chip and its preparation method and application |
| US10196639B2 (en) | 2015-10-09 | 2019-02-05 | University Of Southampton | Modulation of gene expression and screening for deregulated protein expression |
| CN109994154A (en) * | 2017-12-30 | 2019-07-09 | 安诺优达基因科技(北京)有限公司 | A kind of screening plant of single-gene recessive genetic disorder candidate disease causing genes |
| CN107034285B (en) * | 2017-05-17 | 2019-09-24 | 中南大学湘雅医院 | Gene chip for hereditary leukoencephalopathy and its application |
| US10683503B2 (en) | 2017-08-25 | 2020-06-16 | Stoke Therapeutics, Inc. | Antisense oligomers for treatment of conditions and diseases |
| CN112043722A (en) * | 2020-09-25 | 2020-12-08 | 中国医科大学 | Use of PRPF6 in the treatment of prostate cancer and castration resistant prostate cancer |
| US11083745B2 (en) | 2015-12-14 | 2021-08-10 | Cold Spring Harbor Laboratory | Antisense oligomers for treatment of autosomal dominant mental retardation-5 and Dravet Syndrome |
| US11096956B2 (en) | 2015-12-14 | 2021-08-24 | Stoke Therapeutics, Inc. | Antisense oligomers and uses thereof |
| CN115083516A (en) * | 2022-07-13 | 2022-09-20 | 北京先声医学检验实验室有限公司 | Panel design and evaluation method for detecting gene fusion based on targeted RNA sequencing technology |
| US11814622B2 (en) | 2020-05-11 | 2023-11-14 | Stoke Therapeutics, Inc. | OPA1 antisense oligomers for treatment of conditions and diseases |
| US12060558B2 (en) | 2018-05-04 | 2024-08-13 | Stoke Therapeutics, Inc. | Methods and compositions for treatment of cholesteryl ester storage disease |
| CN119320817A (en) * | 2024-10-31 | 2025-01-17 | 迈杰转化医学研究(苏州)有限公司 | Preparation method and application of quality control product or reference product for HER2 gene amplification detection |
-
2013
- 2013-01-07 CN CN201310005252.9A patent/CN103667438B/en not_active Expired - Fee Related
- 2013-01-07 CN CN201410047797.0A patent/CN104004826B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| KONSTANTINOS NIKOPOULOS: "Next-Generation Sequencing of a 40 Mb Linkage Interval Reveals TSPAN12 Mutations in Patients with Familial Exudative Vitreoretinopathy", 《THE AMERICAN JOURNAL OF HUMAN GENETICS》 * |
| MARI´A GONZA´ LEZ-DEL POZO: "Mutation Screening of Multiple Genes in Spanish Patients with Autosomal Recessive Retinitis Pigmentosa by Targeted Resequencing", 《PLOS ONE》 * |
| SARA J. BOWNE: "Mutation Screening of Multiple Genes in Spanish Patients with Autosomal Recessive Retinitis Pigmentosa by Targeted Resequencing", 《BIOCHEMISTRY AND MOLECULAR BIOLOGY》 * |
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| CN115083516A (en) * | 2022-07-13 | 2022-09-20 | 北京先声医学检验实验室有限公司 | Panel design and evaluation method for detecting gene fusion based on targeted RNA sequencing technology |
| CN119320817A (en) * | 2024-10-31 | 2025-01-17 | 迈杰转化医学研究(苏州)有限公司 | Preparation method and application of quality control product or reference product for HER2 gene amplification detection |
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| CN104004826A (en) | 2014-08-27 |
| CN103667438B (en) | 2015-04-01 |
| CN104004826B (en) | 2016-03-02 |
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