CN109055547A

CN109055547A - The biomarker of one group of assessment dissection of aorta risk and its application

Info

Publication number: CN109055547A
Application number: CN201811161226.4A
Authority: CN
Inventors: 王赞鑫; 魏民新
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-09-30
Filing date: 2018-09-30
Publication date: 2018-12-21
Anticipated expiration: 2038-09-30
Also published as: CN114891873A; CN109055547B; CN115029430A

Abstract

The invention discloses the biomarker of one group of assessment dissection of aorta risk, the combination of the biomarker one or more gene mutation sites in table 1.Further, the present invention provides application of the gene mutation site in the testing product of preparation detection or assessment dissection of aorta disease.The present invention is by having found the susceptible mutational site of 44 dissection of aorta and having studied it in the application prospect of dissection of aorta early prediction, the relevant gene loci of dissection of aorta provided by the invention can be used as the early stage biomarker of dissection of aorta, for the genetic molecule mechanism for further studying dissection of aorta, the drug target for exploring dissection of aorta early prevention and treatment provides new direction.

Description

The biomarker of one group of assessment dissection of aorta risk and its application

Technical field

The present invention relates to biomedicines to lead detection technique field, and in particular to the biology of one group of assessment dissection of aorta risk Marker and its application.

Background technique

Dissection of aorta (Aortic Dissection, AD) means aorta under a series of effect of external factors (such as height The factors such as blood pressure, wound), based on aorta wall itself presence or absence of lesion, there is cut in aortic tunica intima, and blood is autonomous Endarterium cut invades the middle layer of aorta wall, and medial constantly tears, is longitudinally separated, causes to show a kind of active State vessel lumen true and false two chamber and deposited.Dissection of aorta onset is hurried, quickly grows, and prognosis is dangerous.Although current is various Treatment means are continuously improved, but the morbidity and mortality of AD are still high, and if without appropriate and timely Treatment, the AD death rate is high.It has been reported that there are about nearly 20% AD patients before reaching hospital is dead, and 30% patient It is dead during hospitalization.Effective pharmaceutical intervention means clinically are lacked to AD at present, are mainly carried out using surgical operation Vascular replacement, intervention operation carry out aorta intra-cavity stent implantation and the two connected applications, but operation risk is big, expense Height, and recurrence rate and re-treatm ent rate at a specified future date is still higher.Along with the progress of Imaging Technology and Medical Devices condition, AD Disease detection rate is in rising trend, and the research of generation and pathogenesis for AD is also deepening continuously.AD generting machanism and something lost It is closely related to pass.AD correlation tumor susceptibility gene is current AD genetics research hot spot, relevant to AD easily from gene angle analysis It is meaningful to feel gene.

The development of plan completion and high throughput sequencing technologies, the research work of medical domain are sequenced along with human genome Person to human genome have deeper into understanding, function and regulated and control network to disease correlative heritability, related gene Understanding is also gradually deepened, and high-flux sequence is used widely.Full sequencing of extron group (Whole Exome Sequencing, WES) technology is to capture entire exon 1 domain dna using exon trapping kit, then carries out high-flux sequence, is current Highly developed a part in genomics research.Over the past two years, started using the document report of WES technical research complex disease Occur, shows the huge advantage of the technology.The scholars such as Ziganshin BA carry out 102 aneurysm of thoracic aorta and AD patient WES analysis, 21.6% patient is there are the mutation of one or more other genes for discovery, 3.9% patient there are FBN1, COL5A1, The detrimental mutation of MYLK, FLNA gene.Janice L.Farlow etc. has carried out WES, discovery 68 to 45 intracranial aneurysm patients There is variation in a gene, the expression of TMEM132B gene is dramatically different in patient and collator (44VS 16).Show common Rare variation can be identified in complex disease by high-flux sequence.Also, there is increasing evidence that rare variation is to normal See disease susceptibility have in arrive intensity effects, significant portion of genetic defect can be explained.It is rare mutation (MAF < 1% or 5%) limitation, and expense, time cost are higher.Whole-genome association (genome wide association Studies, GWAS) can only identify common variation (common variants, CV:MAF > 5%), have ignored it is certain it is unknown with The relevant function candidate gene of AD morbidity.WES compared to genome sequencing (Whole Genome Sequencing, WGS), GWAS, which has, is sequenced the high depth in genome encoding region, in addition to the missing of large fragment repeats and is mutated, moreover it is possible to find low frequency Mutation, rare mutation, identification range is wider, has low cost, efficient advantage, is to find diseases predisposing gene or site spy It is not one of the new tumor susceptibility gene of discovery or the most common method in site, can make up GWAS cannot screen lacking for unknown candidate gene It falls into.

AD vascular risk is high, it is not easy to which early detection, the genetic diagnosis positive rate based on known Disease-causing gene is limited, difficult In doing early stage risk assessment, it would be highly desirable to find new molecular marker.

Summary of the invention

To solve the above-mentioned problems, the purpose of the present invention is to provide the biological markers of one group of assessment dissection of aorta risk Object, the combination of the biomarker one or more gene mutation sites in table 1.

Further, the present invention provides the gene mutation site in the reagent of preparation detection dissection of aorta disease Application.

Further, the present invention also provides the gene mutation sites in preparation assessment dissection of aorta disease risks Application in detection kit.

Further, the present invention provides the gene mutation site in preparation detection dissection of aorta disease device Application.

Preferably, the detection device is sequence testing chip.

Further, the present invention provides a kind of detection kits for assessing dissection of aorta risk comprising detection The reagent whether gene mutation site described in table 1 mutates.

Preferably, the reagent include the primer of gene mutation site described in specific amplification table 1, dNTPs, Taq enzyme, Mg²⁺With PCR reaction buffer.

Preferably, the kit can be the reagent being mutated using any technology detection known in the art, as long as its Gene mutation site in sample can be detected, and, with the presence or absence of mutation, the gene mutation site is as shown in table 1.It includes but not It is limited to each embodiment being set forth below.

With IPO4_c.1342C > G_p.P448A in table 1 and CCDC88B_c.1307C > G_p.S436C gene mutation site For.

In the first embodiment, the kit is including the use of relevant to dissection of aorta prominent in PCR sequencing PCR detection sample Conjugate the presence of the site point IPO4_c.1342C > G_p.P448A G allele and/or susceptible mutational site CCDC88B_ C.1307C reagent existing for the > site G_p.S436C G allele.PCR sequencing PCR is technology known in the field, and required draws The reagents such as object, those of ordinary skill in the art can be voluntarily selected as needed (referring to the companies such as ABI, Beckman sequenator phase Close operation instruction), details are not described herein.Using the kit, by PCR sequencing PCR can directly measure in sample with aorta clamp The sequence in the relevant IPO4_c.1342C > G_p.P448A of layer and/or the mutational site CCDC88B_c.1307C > G_p.S436C, from And judge whether it carries the variation of corresponding site allele, and then judge the neurological susceptibility of its dissection of aorta.

In this second embodiment, the kit including the use of Taqman probe abrupt climatic change method detection sample in master The relevant mutational site IPO4_c.1342C > G_p.P448A of artery dissection and/or CCDC88B_c.1307C > G_p.S436C base Because of the reagent of type.Wherein Taqman probe used is for IPO4_c.1342C > G_p.P448A and/or CCDC88B_c.1307C The probe of Reagent Company's offer can be used in the probe of the mutational site > G_p.S436C design, the probe；Software can also be used certainly Row design, such as the Beacon Designer 7.5 of PREMIERBiosoft company.

In the third embodiment, the kit is to detect IPO4_ in sample using PCR- Single strand conformation polymorphism C.1342C the kit of > G_p.P448A and/or the mutational site CCDC88B_c.1307C > G_p.S436C genotype.The reagent It include for expanding IPO4_c.1342C > G_p.P448A and/or the mutational site CCDC88B_c.1307C > G_p.S436C in box Primer, PCR reagent, check sample and detect conformation electrophoresis needed for reagent.The preferred non-denaturing polyacrylamide of electrophoresis Gel electrophoresis.It include the homozygous negative control sample of mutational site IPO4_c.1342C > G_p.P448A CC in check sample It can also include simultaneously in addition susceptible mutational site at least one of with the homozygous positive control of site GG The homozygous negative control sample of CCDC88B_c.1307C > G_p.S436C CC and the homozygous positive control of site GG At least one of, it additionally may include the check sample that can not also include the corresponding heterozygote of heterozygote.It is preferred that wrapping simultaneously Include above-mentioned three classes check sample.The amplified production of sample to be tested amplified production and check sample electrophoresis simultaneously, compares its electrophoresis knot Whether fruit is it can be concluded that carry the testing result of corresponding allelic variation in sample to be tested.

Still further, the present invention provides a kind of screening sides in screening mutational site relevant to dissection of aorta disease Method the described method comprises the following steps:

(1) DNA of the peripheral blood sample of dissection of aorta patient and normal control is extracted；

(2) DNA is subjected to ultrasonic fragmentation, interrupts fragment ends filling-in, 3 ' ends plus A, connects adaptor, select 350~ Segment between 400bp prepares full-length genome library；

(3) full exon detection is carried out using GenCap liquid phase capture target gene technology；It is carried out with high-flux sequence instrument Both-end sequencing, reads a length of 100bp；

(4) after exon sequencing, conventional filtration analysis is carried out, is with reference to genome version with hg19, db mutation (v147) For screening and filtering standard；Pathogenic sites, normal person's frequency are left 5% hereinafter, and having same sense mutation reported in the literature to cause a disease position Point；In order to more accurately screen disease related locus, it is pre- to filter out SIFT, Polyphen2, MutationTaster, GERP++ Survey is all benign mutation；Filter out Mutcount > 5, MutRatio > 30%, the Indel of MAF > 1% in normal data library；

(5) the exclusion work in false positive site is carried out, first is that by IGV software to the bam after de-redundancy duplicates File (rmdup.sorted.bam) carries out checking verifying, and catastrophe is incongruent to be considered as false positive site；Pass through simultaneously Samtools software checks bam file.Then, respectively with the gene (a) of mutation, mutation site (b) as Maker carries out rare mutation load (RVB) analysis, the sample number of the gene mutation and calculating in statistics case group and control group OR value, OR>1, P<0.01.As a result, it has been found that thering are multiple mutational sites and AD to have statistical correlations；

(6) depth is carried out to sequencing result by gene function and the analysis of the annotation of related pathways and literature search data Analysis, final inventor filter out 44 mutational sites relevant to dissection of aorta.

The invention has the advantages that:

The present invention is by having found 44 gene mutation sites relevant to dissection of aorta and having studied it in aorta The application prospect of interlayer early prediction, the relevant gene mutation site of dissection of aorta provided by the invention can be used as aorta clamp It is anti-to explore dissection of aorta early stage for the genetic molecule mechanism of further research dissection of aorta for the early stage biomarker of layer The drug target controlled provides new direction.

Detailed description of the invention

It includes two or more base in access that 44 site GO path analysis of Fig. 1 (Molec μ Lar Function), which are chosen, Cause, p value are less than or equal to 0.05 access, to enrichment score mapping；

It includes two or more base in access that 44 site GO path analysis (biological process) of Fig. 2, which are chosen, Cause, p value are less than or equal to 0.05 access, to enrichment score mapping.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment Used in the conventional means that are well known to those skilled in the art of technological means, reagent used can be commercially available.

Test method without specific conditions in embodiment, usually conventional method in that art, such as according to normal conditions Such as Sambrook et al., molecular cloning, the condition in laboratory manual (third edition) (Science Press, 2002), or according to Condition proposed by reagent manufacturing firm.

1 sample collection of embodiment

It is made a definite diagnosis from January, 2017 in April, 2018 in Shenzhen's sun yat-sen angiocardiopathy hospital by aorta CTA 99 patients of Stanford A type dissection of aorta collect whole blood 2mL and 590 normal control samples and are sequenced from Mai Jinuo The database of company's retrieval.The informed consent of patient has been obtained, and has been audited by Ethics Committee.

Sample process: EDTA anticoagulated whole blood is mixed in 1:1 ratio with Trizol, mixes well and is placed on 1.8mL cell In cryopreservation tube, the refrigerator preservation that 30s is placed on -80 DEG C is cooled down rapidly in liquid nitrogen.

Embodiment 2 extracts blood sample DNA

(1) 1mL is passed through and is added in the blood of EDTA (0.01M, China, Huamei Bioengineer) anticoagulation 1mLCL cell pyrolysis liquid is gently mixed by inversion 6 times, is centrifuged 5min, reject supernatant with the revolving speed of 3600rpm；

(2) it as pouring into 1mLCL cell pyrolysis liquid in centrifuge tube again, is gently mixed by inversion 6 times, with turning for 3600rpm Speed centrifugation 5min, reject supernatant；Under the premise of ensuring that precipitating is retained in pipe, centrifuge tube is upside down on clean blotting paper Stand 2min；

(3) mixed liquor of Proteinase K and buffer FG is configured；

(4) mixed liquor of 500 μ L Proteinase Ks and buffer FG is added, is mixed immediately to solution without agglomerate；

(5) 65 DEG C of water-bath 30min, are during which mixed by inversion for several times；

(6) 1mL isopropanol is added, is mixed by inversion immediately to there is tufted or Filamentous genomic DNA；

(7) 8min, reject supernatant are centrifuged with the revolving speed of 3600rpm；It, will be under the premise of ensuring to precipitate and being retained in pipe Heart pipe is upside down on clean blotting paper and stands 2min；

(8) 1mL70% ethyl alcohol is added, vibrates 5sec, is centrifuged 3min, reject supernatant with the revolving speed of 3600rpm；

(9) step (8) are repeated；

(10) under the premise of ensuring that precipitating is retained in pipe, centrifuge tube is upside down on clean blotting paper and stands 5min；

(11) under normal temperature state, be air-dried gene group DNA be precipitated to all liq volatilize completely (at least 5min) oscillation 5sec is centrifuged 3min, reject supernatant with the revolving speed of 3600rpm；

(12) 200 μ LTB buffers are added, low speed vortex vibrates 5sec, during which 65 DEG C of heating water bath 1h flick hydrotropy number It is secondary；

(13) extracted genomic DNA concentration and purity are identified using NanoDrop ND8000 (THERMO, USA).It is dense > 30ng/ μ L, 1.8 < OD260/OD280 < 2.0 are spent, and the genomic DNA sample of 3 μ g of total amount > is considered as qualification, is stored in- 80 DEG C of refrigerator is spare；

(14) on the preliminary quantitative basis of DNA, further using agarose gel electrophoresis detection (gum concentration: 0.8%, electricity Pressure: 120V, time: 20min) accurate quantification DNA.Occur illustrating that DNA may have there are miscellaneous band except weak band or master tape after electrophoresis Signs of degradation or there are impurity illustrates that the DNA mass extracted is higher if occurring a strong band after electrophoresis.Full exon is surveyed Sequence requires DNA concentration >=50ng/ μ L, and total amount is not less than 5 μ g, OD260/280 between 1.8~2.0, and OD260/230 is on 2.0 left sides The right side, sample are polluted without RNA, no degradation or slight degradation.

The sequencing of the full exon of embodiment 3

1, DNA library constructs

3 μ g DNA are subjected to ultrasonic fragmentation, fragment ends filling-in, 3 ' ends plus A is interrupted, connects adaptor, select 350 Segment between~400bp prepares full-length genome library.Library sample uses Agilent2100 biological analyser (Agilent section Skill company, the U.S.) carry out Quality Control.

2, target area capture sequencing

Full exon detection is carried out using GenCap liquid phase capture target gene technology (Beijing Mai Jinuo company).By 1 μ g DNA library is mixed with BL buffer and probe, and 95 DEG C of heating 7min, the HY that 65 DEG C of 23 μ L of heating 2min addition are preheated to 65 DEG C are slow Fliud flushing, 65 DEG C of hybridization 22h.Cleaning 50 μ L MyOne magnetic beads with 500 μ L 1X combination buffers, (U.S. Life Technology is public Department) 3 times, it is resuspended in 80 μ L 1X combination buffers.64 μ L 2X combination buffers are added into hybridization mixture, be transferred to containing In the test tube of 80 μ L MyOne magnetic beads.Rotation mixes 1h.Magnetic bead 15min is cleaned with WB1 buffer room temperature, with WB3 buffer 65 DEG C cleaning 3 times, each 15min.Then it is eluted with the combining DNA of elution buffer.The DNA of elution carries out PCR reaction, Reaction condition is as follows: 98 DEG C of initial denaturation 30s；98 DEG C of denaturation 25s, 65 DEG C of annealing 30s, 72 DEG C of extension 30s carry out 15 altogether and follow Ring；Last 72 DEG C of extensions 5min.PCR is purified with SPRIbeads (U.S. Beckman Co μ Lter) according to product description to produce Object.The library of enrichment carries out both-end sequencing with 2000 sequenator of illumina HiSeq, reads a length of 100bp.

3, data are analyzed

Obtained primitive sequencer sequence is sequenced, the inside, can be to follow-up point containing belt lacing, low-quality reads Analysis interferes.In order to guarantee information analysis quality, initial data is filtered with Cutadapt software, connector sequence will be removed Column and the sequencing data of low quality base are compared with Burrows-Wheeler Aligner (BWA) arrives human genome (version GRCh37/hg19 on), and redundancy is removed with Picard tool.The depth of target area is calculated with coverageBed tool.It is logical GATK software is crossed indel location proximate is compared again to improve the quality of sequence.GATK HaplotypeCaller inspection Mutation and indel abrupt information are surveyed, and mutation is annotated with ANNOVAR, annotations database includes db mutation 147,1000 Genomes project,Exome sequencing project(ESP6500),Inhouse database (MyGenostics),gnomAD_genome_EAS.Online Mendelian Inheritance in Man(OMIM)and Human Gene Mutation Database(HGMD Professional 2016.10).Pathogenic forecast database includes SIFT, PolyPhen-2 and MutationTaster, and divided by ACMG standard guide to pathogenic.

4, variation filtering

In order to find out potential disease cause mutation, need to be filtered data screening.Mainly for exon 1 and can Become the mutation of share zone, step 1: leaving the disease cause mutation in pathogenic analysis (pathogenic_analysis) (pathogenic) site.Filter screen criteria is as follows:

Step 2: screening mutating alkali yl sequencing number be greater than 5, the frequency of mutation be more than or equal to 30% and Retain in five normal person's mutation databases of 1000g2015apr, ESP6500si, Inhouse, ExAC_ALL, ExAC_EAS and does not have It occurs or the site less than 5%；The same sense mutation site in data set is removed, in addition has mutational site reported in the literature to stay Under.

5, it statisticallys analyze

Rare mutation load Analysis is carried out to above-mentioned filtered mutation result, selects 590 number of cases of Mai Jinuo according to as right In the same old way this, using the Metabin analysis OR value and 95% confidence interval in R language, and with Mantel-Haenszel ' s method into Row statistical check, p value think significant less than 0.01.

6, result

After exon sequencing, conventional filtration analysis is carried out, screening and filtering standard: being reference with hg19, db mutation (v147) Genome version.The pathogenic site (pathogenic), normal person's frequency are left 5% hereinafter, and having reported in the literature synonymous prominent Become pathogenic sites, obtains 59351 point mutation.In order to more accurately screen disease related locus, further filter out SIFT, Polyphen2, MutationTaster, GERP++ prediction are all benign mutation；Filter out Mutcount > 5, MutRatio > 30%, the Indel of MAF > 1% in normal data library；29979 point mutation are obtained through above-mentioned screening.

Later, inventor has carried out the exclusion work in false positive site, first is that by IGV software to de-redundancy Bam file (rmdup.sorted.bam) after duplicates carries out checking verifying, and catastrophe is incongruent to be considered as false sun Property site；Bam file is checked by samtools software simultaneously.Then, respectively with the gene (a) of mutation, mutation Site (b) carries out rare mutation load (RVB) analysis as maker, the sample of the gene mutation in statistics case group and control group This number simultaneously calculates OR value, OR>1, P<0.01.As a result, it has been found that thering are 44 mutational sites and AD to have statistical correlations, as shown in table 1.

The gene mutation site relevant to dissection of aorta of table 1

The annotation of embodiment 4 gene function and related pathways

Inventor carry out to 44 mutational sites filtered out the annotation of gene function and related pathways.

GO (Gene Ontology): describing the comprehensive library of gene function, can be divided into molecular function (Molec μ lar Function, MF), bioprocess (biological process, BP) and cell composition (cell μ l ar component, CC) three parts.GO enrichment is significant enrichment less than 0.05 with p.

The mapping that count is more than or equal to 2 is chosen in the access annotation of gene where 44 mutational sites, as shown in Figure 1, 2, In BP (biological process, bioprocess) analysis, gene significant enrichment is raw in redox where these sites Object process.

It is found in the selection result in 44 mutational sites:

PTGR1_c.655G > A_p.G219S (OR=55.65,95%CI=2.97~1041.89), HSDL2_c.593A > C_p.D198A (OR=24.8,95%CI=2.74~224.27), CBR4_c.-87_-88delTT_- (OR=3.9,95%CI It=1.57~9.67) is the relevant site of oxidation-reduction process.

BID_c.214C > T_p.R72C (OR=24.8,95%CI=2.74~224.27), DAZAP1_c.* 276delG_- (OR=12.38,95%CI=2.24~68.52), SETMAR_c.1025G > T_p.S342I (OR=7.55, 95%CI=2.26~25.23) relevant to cell migration differentiation site mutation.The proliferation of vascular endothelial cell, migration, differentiation It can lead to blood vessel endothelium metamorphosis, it may be in the endangium metamorphosis correlation of AD.Separately there is LAD1_c.430C > T_ P.R144W (OR=8.24,95%CI=1.82~37.39) is related to cadherin, and Vascular Endothelial Cadherin with Arterial disease has certain relevance.

CYBRD1_c.293T > C_p.M98T (OR=31.33,95%CI=3.62~271.13), PCDH12_c.728C > T_p.A243V (OR=12.38,95%CI=2.24~68.52), ZNF717_c.1498A > G_p.T500A (OR=4.16, 95%CI=1.45~11.97) it is then related in conjunction with metal ion.Metal ion and antiotasis have close ties, blood vessel Power may exist with AD and contact by influencing blood pressure.

PLA2G7_c.*81_*78delTGTG_- (OR=38,95%CI=4.52~319.21), ITCH_c.385A > G_ P.T129A (OR=15.64,95%CI=2.99~81.77), PKHD1_c.11525G > A_p.R3842Q (OR=12.38, 95%CI=2.24~68.52), CAMP_c.154C > T_p.R52W (OR=8.24,95%CI=1.82~37.39) may be with The processes such as lipid-metabolism, response to oxidative stress, inflammatory reaction, platelet activating factor metabolism are related.

Embodiment 5 is used to predict the production of dissection of aorta disease early diagnosis kit

The kit of the present invention for dissection of aorta is assembled, the kit may include specific amplification table The primer kit of one or more gene locis in 1.

Specifically there are following 4 kinds of schemes:

Kit 1: nucleosides of the kit including the site specific amplified CCDC88B_c.1307C > G_p.S436C The primer pair of acid sequence is as shown in SEQ ID NO:1 and SEQ ID NO:2.

Kit 2: nucleotide of the kit including the site specific amplified IPO4_c.1342C > G_p.P448A The primer pair of sequence is as shown in SEQ ID NO:3 and SEQ ID NO:4.

Kit 3: nucleotides sequence of the kit including the site specific amplified CPNE9_c.260T > C:p.V87A The primer pair of column is as shown in SEQ ID NO:5 and SEQ ID NO:6.

Kit 4: nucleosides of the kit including the site specific amplified CCDC88B_c.1307C > G_p.S436C As shown in SEQ ID NO:1 and SEQ ID NO:2, the amplification site IPO4_c.1342C > G_p.P448A exists the primer pair of acid sequence The primer pair of interior nucleotide sequence is as shown in SEQ ID NO:3 and SEQ ID NO:4, and amplification CPNE9_c.260T > C: The primer pair of nucleotide sequence including the site p.V87A is as shown in SEQ ID NO:5 and SEQ ID NO:6.

The amplimer is as follows:

CCDC88B_c.1307C>G_p.S436C:

SEQ ID NO:15’-GCTGGCTGAGGAGAATGTGG-3’,

SEQ ID NO:25'-TGAGGGCTGTGGTCGAAGG-3'；

IPO4_c.1342C>G_p.P448A:

SEQ ID NO:35’-CCATATCAGCAGCTATTCAAGG-3’,

SEQ ID NO:45'-CGCAAGTCAGGGTCGTCTA-3'；

CPNE9_c.260T>C:p.V87A:

SEQ ID NO:55’-CCCGGCCACCAAGATTGAA-3’,

SEQ ID NO:65’-GTGAGGGTTCGCTCTACTCG-3’。

Reagent needed for the kit further includes PCR reagent, check sample and detects the electrophoresis of conformation.Needed for round pcr Common agents, such as: dNTPs, MgCl₂, distilled water, Taq enzyme etc..The preferred non-denaturing polyacrylamide gel electricity of electrophoresis Swimming.In addition it can have standard items and control (such as determining standard items and the blank control of genotype).Sample to be tested amplification produces The amplified production of object and check sample electrophoresis simultaneously compares its electrophoresis result it can be concluded that whether carrying in sample to be tested corresponding etc. The testing result of position genetic mutation.

The value of this kit is only to need peripheral blood without other tissue samples, by most simplifying and special Primer pair detection mutation, then pass through spectrum of mutation auxiliary judgment dissection of aorta, it is not only stable, easy to detect and accurate, it mentions significantly The sensibility and specificity of high medical diagnosis on disease, therefore this kit is put into and is practiced can help to instruct diagnosis and more effectively Individualized treatment.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

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Claims

1. the biomarker of one group of assessment dissection of aorta risk, which is characterized in that the biomarker is one in table 1 The combination of a or multiple gene mutation sites.

2. application of the gene mutation site described in claim 1 in the reagent of preparation detection dissection of aorta disease.

3. gene mutation site described in claim 1 is in the detection kit of preparation assessment dissection of aorta disease risks Using.

4. application of the gene mutation site described in claim 1 in preparation detection dissection of aorta disease device.

5. application as claimed in claim 4, which is characterized in that the detection device is sequence testing chip.

6. a kind of detection kit for assessing dissection of aorta risk, which is characterized in that it includes that gene described in detection table 1 is prominent The reagent whether displacement point mutates.

7. detection kit as claimed in claim 6, which is characterized in that the reagent includes described in specific amplification table 1 Gene mutation site primer, dNTPs, Taq enzyme, Mg²⁺With PCR reaction buffer.

8. a kind of screening technique for screening mutational site relevant to dissection of aorta disease, which is characterized in that the method packet Include following steps:

(3) full exon detection is carried out using GenCap liquid phase capture target gene technology；Both-end is carried out with high-flux sequence instrument Sequencing, reads a length of 100bp；

(4) after exon sequencing, conventional filtration analysis is carried out, is sported with reference to genome version using hg19, db as screening and filtering Standard；Pathogenic sites, normal person's frequency are left 5% hereinafter, and there are a same sense mutation pathogenic sites reported in the literature；In order to more quasi- True screening disease related locus filters out SIFT, Polyphen2, MutationTaster, GERP++ prediction and is all benign Mutation；Filter out Mutcount > 5, MutRatio > 30%, the Indel of MAF > 1% in normal data library；

(5) the exclusion work in false positive site is carried out, first is that by IGV software to the bam file after de-redundancy duplicates It carries out checking verifying, catastrophe is incongruent to be considered as false positive site；Bam file is carried out by samtools software simultaneously It checks；Then, respectively with the gene of mutation, mutation site as maker, carry out rare mutation load Analysis, count case It organizes the sample number with the gene mutation in control group and calculates OR value, OR>1, P<0.01；As a result, it has been found that have multiple mutational sites with AD has statistical correlations；

(6) depth point is carried out to sequencing result by gene function and the analysis of the annotation of related pathways and literature search data Analysis, final inventor filter out 44 mutational sites relevant to dissection of aorta.