CN109097466B - SNP (Single nucleotide polymorphism) site related to aortic dissection disease and application thereof - Google Patents

Abstract

The invention discloses a method for screening SNP sites related to aortic dissection disease based on whole-exon sequencing technology. The SNP sites include one or two of the following aortic dissection susceptibility SNP sites: PTK6_c. 205G>A_p.E69K; TSHZ2_c.724C>T_p.R242C. Further, the present invention provides the application of the SNP site in a detection product for detecting aortic dissection disease. The present invention discovers two aortic dissection susceptible SNP loci and studies their application prospects in early prediction of aortic dissection, and the aortic dissection-related gene loci provided by the present invention may be early biomarkers of aortic dissection It provides a new direction for further studying the genetic molecular mechanism of aortic dissection and exploring drug targets for early prevention and treatment of aortic dissection.

Description

SNP loci associated with aortic dissection disease and its application

technical field

The invention relates to the technical field of biomedical detection, in particular to a SNP site related to aortic dissection disease and its application.

Background technique

Aortic dissection (AD) refers to the rupture of the aortic intima under the action of a series of external factors (such as hypertension, trauma, etc.), based on the presence or absence of lesions in the aortic wall itself. The blood aortic intimal rupture invades the middle layer of the aortic wall, and the middle layer of the aorta is continuously torn and separated longitudinally, resulting in a state of coexistence of true and false aortic lumen. The onset of aortic dissection is rapid, the development is rapid, and the prognosis is dangerous. Despite the continuous improvement of various current treatments, the morbidity and mortality of AD remains high, and if not properly and timely treated, AD mortality is extremely high. It has been reported that nearly 20% of AD patients die before reaching the hospital, and 30% die during hospitalization. At present, there is no effective drug intervention for AD in clinical practice. Surgical surgery for vascular replacement, interventional surgery for aortic stent implantation, and the combination of the two are mainly used. However, the surgical risk is high, the cost is high, and the long-term recurrence rate is high. and reoperation rates remain high. With the advancement of imaging technology and medical equipment conditions, the detection rate of AD disease is on the rise, and the research on the occurrence and pathogenesis of AD is also deepening. The mechanism of AD generation is closely related to genetics. AD-related susceptibility genes are the current research hotspots in AD genetics, and it is meaningful to analyze AD-related susceptibility genes from a genetic perspective.

With the completion of the Human Genome Sequencing Project and the development of high-throughput sequencing technology, researchers in the medical field have gained a deeper understanding of the human genome, and have gradually deepened their understanding of disease-related genetic variants, functions of related genes, and regulatory networks. , high-throughput sequencing is widely used. Whole Exome Sequencing (WES) technology uses an exon capture kit to capture the DNA of the entire exon region, and then performs high-throughput sequencing, which is a very mature part of the current genomics research. In the past two years, literature reports using WES technology to study complex diseases have begun to appear, demonstrating the great advantages of this technology. ZiganshinBA et al. performed WES analysis on 102 patients with thoracic aortic aneurysm and AD, and found that 21.6% of patients had mutations in one or more other genes, and 3.9% of patients had deleterious mutations in FBN1, COL5A1, MYLK, and FLNA genes. Janice L. Farlow et al. performed WES on 45 patients with intracranial aneurysms and found that 68 genes had mutations, and the expression of TMEM132B gene was significantly different between patients and controls (44VS 16). Demonstrate that rare variants can be identified by high-throughput sequencing in common complex diseases. Furthermore, there is growing evidence that rare variants have moderate to strong effects on common disease susceptibility, explaining a large proportion of genetic deletions. The limitations of rare mutations (MAF < 1% or 5%), and the cost and time cost are high. Genome wide association studies (GWAS) can only identify common variants (CV: MAF>5%), ignoring some unknown functional candidate genes associated with AD pathogenesis. Compared with Whole Genome Sequencing (WGS) and GWAS, WES has a high-depth sequencing of the coding region of the genome. In addition to large fragments of deletions and duplications and SNPs, it can also find low-frequency mutations and rare mutations, and has a wider recognition range. The advantages of low cost and high efficiency are one of the most commonly used methods to find disease susceptibility genes or loci, especially new susceptibility genes or loci, which can make up for the defect that GWAS cannot screen unknown candidate genes.

AD has a high vascular risk and is not easy to detect early. The positive rate of genetic diagnosis based on known causative genes is limited, making early risk assessment difficult. New molecular markers are urgently needed to be discovered.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned problems, the purpose of the present invention is to provide a method for screening SNP loci related to aortic dissection disease based on whole exome sequencing technology, the SNP locus includes one of the following aortic dissection susceptibility SNP loci One or both: PTK6_c.205G>A_p.E69K; TSHZ2_c.724C>T_p.R242C.

Further, the present invention provides the application of the SNP site in the preparation of a reagent for detecting aortic dissection disease.

Further, the present invention also provides the application of the SNP site in preparing an early diagnosis kit for predicting aortic dissection disease.

Preferably, the kit includes a reagent for detecting whether the aortic dissection susceptibility SNP site PTK6_c.205G>A_p.E69K and/or TSHZ2_c.724C>T_p.R242C is mutated.

Preferably, the kit can be a reagent for detecting SNP using any technology known in the art, as long as it can detect the susceptible SNP site PTK6_c.205G>A_p.E69K and/or TSHZ2_c.724C>T_p. Whether there is a mutation in R242C. It includes, but is not limited to, the various embodiments listed below.

In the first embodiment, the kit includes detecting the presence of the A allele at the susceptible SNP site PTK6_c.205G>A_p.E69K site in the sample by sequencing, and/or the susceptible SNP site TSHZ2_c.724C>T_p .Reagents for the presence of the T allele at the R242C site. The sequencing method is a well-known technology in the art, and the required primers and other reagents can be selected by those of ordinary skill in the art according to their needs (refer to the relevant instructions of sequencers from companies such as ABI and Beckman), which will not be repeated here. Using this kit, the sequence of the susceptible SNP locus PTK6_c.205G>A_p.E69K and/or TSHZ2_c.724C>T_p.R242C in the sample can be directly measured by sequencing, so as to determine whether it carries the corresponding locus allele Variation in the susceptibility to aortic dissection.

In the second embodiment, the kit includes reagents for detecting the genotypes of susceptible SNP sites PTK6_c.205G>A_p.E69K and/or TSHZ2_c.724C>T_p.R242C in the sample using Taqman probe SNP detection method. The Taqman probe used is a probe designed for the susceptible SNP site PTK6_c.205G>A_p.E69K and/or TSHZ2_c.724C>T_p.R242C, the probe can use the probe provided by the reagent company; also can use the software Design by yourself, such as BeaconDesigner 7.5 of PREMIERBiosoft.

In the third embodiment, the kit is for detecting the genotypes of susceptible SNP sites PTK6_c.205G>A_p.E69K and/or TSHZ2_c.724C>T_p.R242C genotypes in a sample by PCR-single-strand conformation polymorphism Reagent test kit. The kit includes primers for amplifying susceptible SNP sites PTK6_c.205G>A_p.E69K and/or TSHZ2_c.724C>T_p.R242C, PCR reagents, control samples and reagents required for electrophoresis to detect conformation. The electrophoresis is preferably native polyacrylamide gel electrophoresis. The control sample includes at least one of the negative control sample of the susceptible SNP site PTK6_c.205G>A_p.E69K GG homozygote and the positive control sample of the site AA homozygote, and may also include the susceptible SNP site TSHZ2_c. 724C>T_p.R242C CC homozygote negative control sample, at least one of the positive control sample of site TT homozygote, and may or may not include the control sample of the corresponding heterozygote of the heterozygote. Preferably, the above three types of control samples are included at the same time. The amplified product of the sample to be tested and the amplified product of the control sample are electrophoresed at the same time, and the results of electrophoresis can be compared to determine whether the sample to be tested carries the corresponding allelic variation.

Further, the present invention provides the application of the SNP site in preparing a device for diagnosing aortic dissection disease.

Preferably, the diagnostic device is a sequencing chip.

Still further, the present invention provides a method for screening SNP sites related to aortic dissection disease, the method comprising the following steps:

(1) Extracting DNA from peripheral blood samples of patients with aortic dissection and normal controls;

(2) ultrasonically fragment the DNA, blunt the ends of the broken fragments, add A at the 3' end, connect the adapter, and select fragments between 350 and 400 bp to prepare a whole genome library;

(3) Use GenCap liquid phase capture target gene technology for whole exon detection; use high-throughput sequencer for paired-end sequencing with a read length of 100bp;

(4) After exon sequencing, perform routine filtering analysis, taking hg19 and dbSNP (v147) as the reference genome version as the screening filtering criteria; leaving the pathogenic locus, the frequency of normal people below 5%, and those reported in the literature Synonymous mutation pathogenic loci; in order to more accurately screen disease-related loci, filter out SNPs predicted to be benign by SIFT, Polyphen2, MutationTaster, GERP++; filter out Mutcount>5, MutRatio>30%, MAF> in normal database 1% Indel;

(5) Eliminate false positive sites. First, check and verify the bam file (rmdup.sorted.bam) after de-redundant duplicates through IGV software. Those that do not meet the mutation conditions are regarded as false positive sites; View the bam file through the samtools software. Then, using the mutated gene (a) and the mutated site (b) as the maker, the rare mutation load (RVB) analysis was performed, and the number of samples with the gene mutation in the case group and the control group was counted and the OR value was calculated, OR >1,P<0.01. It was found that there were multiple mutation sites that were statistically associated with AD;

(6) In-depth analysis of the sequencing results through annotation analysis of gene functions and related pathways and literature search data, the inventors finally screened two aortic dissection susceptibility SNP loci from 48 mutation sites: PTK6_c.205G>A_p .E69K and TSHZ2_c.724C>T_p.R242C.

Beneficial effects of the present invention:

The present invention discovers two aortic dissection susceptible SNP loci and studies their application prospects in early prediction of aortic dissection, and the aortic dissection-related gene loci provided by the present invention may be early biomarkers of aortic dissection It provides a new direction for further studying the genetic molecular mechanism of aortic dissection and exploring drug targets for early prevention and treatment of aortic dissection.

Description of drawings

Figure 1 shows the 48-site GO pathway analysis (MolecμLarFunction) selected pathways including two or more genes, and the pathways with p value less than or equal to 0.05 are plotted against the enrichment score;

Figure 2 is a graph of the enrichment scores of the 48 loci GO pathway analysis (biological process) selected pathways including two or more genes, and the p value is less than or equal to 0.05.

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Terms are explained as follows in the present invention:

GO (Gene Ontology): a comprehensive database describing gene function, which can be divided into three parts: molecular function (Molecμlar Function, MF), biological process (biological process, BP), and cell μl arcomponent (CC). GO enrichment was regarded as significant enrichment with p less than 0.05.

Example 1 Sample Collection

From January 2017 to April 2018, 99 patients with Stanford type A aortic dissection were diagnosed by aortic CTA in Sun Yixian Cardiovascular Hospital of Shenzhen, and 2 mL of whole blood and 590 normal control samples were collected from the database searched by Mykino Sequencing Company . The informed consent of the patients has been obtained and reviewed by the ethics committee.

Sample processing: Mix EDTA anticoagulated whole blood with Trizol at a ratio of 1:1, mix well and place in a 1.8mL cell cryopreservation tube, quickly cool in liquid nitrogen for 30s, and store in a -80°C refrigerator.

Example 2 Extraction of DNA from blood samples

(1) Add 1 mL of CL cell lysate to 1 mL of blood anticoagulated by EDTA (0.01M, China, Huamei Bioengineer), gently invert and mix 6 times, centrifuge at 3600 rpm for 5 min, and discard the supernatant;

(2) Pour 1 mL of CL cell lysate into the centrifuge tube again, gently invert and mix 6 times, centrifuge at 3600 rpm for 5 minutes, and discard the supernatant; on the premise of ensuring that the precipitate remains in the tube, invert the centrifuge tube in Let stand on clean absorbent paper for 2min;

(3) configure the mixture of proteinase K and buffer FG;

(4) Add 500 μL of the mixture of proteinase K and buffer FG, and then mix until the solution has no clumps;

(5) 65℃ water bath for 30min, invert and mix several times during this period;

(6) Add 1 mL of isopropanol, then invert and mix until clustered or filamentous genomic DNA appears;

(7) Centrifuge at 3600rpm for 8min, discard the supernatant; under the premise of ensuring that the precipitate remains in the tube, invert the centrifuge tube on clean absorbent paper and let stand for 2min;

(8) Add 1 mL of 70% ethanol, shake for 5 sec, centrifuge at 3600 rpm for 3 min, and discard the supernatant;

(9) Repeat step (8);

(10) Under the premise of ensuring that the precipitation remains in the tube, invert the centrifuge tube on clean absorbent paper and let stand for 5min;

(11) Under normal temperature, air-dry the genomic DNA precipitation until all the liquid is completely volatilized (at least 5min), shake for 5sec, centrifuge at 3600rpm for 3min, and discard the supernatant;

(12) Add 200 μL of TB buffer, vortex at low speed for 5 sec, heat in a water bath at 65°C for 1 h, and flick several times to aid dissolution;

(13) The concentration and purity of the extracted genomic DNA were identified using NanoDrop ND8000 (THERMO, USA). Genomic DNA samples with concentration > 30ng/μL, 1.8 < OD260/OD280 < 2.0, and total amount > 3 μg are considered qualified and stored in a refrigerator at -80°C for later use;

(14) On the basis of preliminary DNA quantification, DNA was further accurately quantified by agarose gel electrophoresis detection (gel concentration: 0.8%, voltage: 120V, time: 20min). If a weak band appears after electrophoresis or there is a secondary band outside the main band, it means that the DNA may be degraded or there are impurities. If a strong band appears after electrophoresis, it means that the extracted DNA is of high quality. Whole exome sequencing requires DNA concentration ≥50ng/μL, total amount not less than 5μg, OD260/280 between 1.8 and 2.0, OD260/230 around 2.0, and the sample has no RNA contamination, no degradation or slight degradation.

Example 3 Whole exome sequencing

1. DNA library construction

3 μg of DNA was fragmented by ultrasonic, the ends of the fragments were blunted, A was added at the 3' end, and the adaptor was connected, and fragments between 350 and 400 bp were selected to prepare a whole genome library. The library samples were quality controlled using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA).

2. Target region capture and sequencing

Whole exome detection was performed using GenCap liquid phase capture target gene technology (Beijing Mykino Co., Ltd.). Mix 1 μg of DNA library with BL buffer and probe, heat at 95 °C for 7 min, and heat at 65 °C for 2 min, add 23 μL of HY buffer preheated to 65 °C, and hybridize at 65 °C for 22 h. 50 μL of MyOne magnetic beads (Life Technology, USA) were washed 3 times with 500 μL of 1X binding buffer, and resuspended in 80 μL of 1X binding buffer. Add 64 μL of 2X Binding Buffer to the hybridization mixture and transfer to a tube containing 80 μL of MyOne Magnetic Beads. Rotate and mix for 1 h. The magnetic beads were washed with WB1 buffer at room temperature for 15 min, and washed with WB3 buffer at 65°C for 3 times for 15 min each time. The bound DNA is then eluted with elution buffer. The eluted DNA was subjected to PCR reaction under the following conditions: pre-denaturation at 98°C for 30s; denaturation at 98°C for 25s, annealing at 65°C for 30s, extension at 72°C for 30s, a total of 15 cycles; and a final extension at 72°C for 5 min. PCR products were purified using SPRIbeads (Beckman Co μLter, USA) according to the product instructions. The enriched library was paired-end sequenced with illumina HiSeq 2000 sequencer with a read length of 100 bp.

3. Data analysis

The original sequence obtained by sequencing contains low-quality reads with adapters, which will interfere with subsequent information analysis. In order to ensure the quality of information analysis, the original data were filtered with Cutadapt software, and the sequencing data with adapter sequences and low-quality bases removed were aligned to the human genome (version GRCh37/hg19) with the Burrows-Wheeler Aligner (BWA), and Picard Tools to remove redundancy. The depth of the target area is calculated with the coverageBed tool. The quality of the sequence was improved by re-aligning near the indel site by GATK software. GATK HaplotypeCaller detects SNP and indel mutation information, and uses ANNOVAR to annotate mutations. Annotation databases include dbSNP147, 1000Genomes 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). The pathogenicity prediction database includes SIFT, PolyPhen-2 and MutationTaster, and the pathogenicity is divided by ACMG standard guidelines.

4. Variation filtering

To identify potential pathogenic variants, the data needs to be filtered. Mainly for mutations in exon regions and alternative splicing regions, the first step: leaving pathogenic mutation sites in pathogenic analysis (pathogenic_analysis). The filtering criteria are as follows:

Step 2: Screen for mutation bases with more than 5 sequencing times, mutation frequency greater than or equal to 30%, and 1000g2015apr, ESP6500si, Inhouse, ExAC_ALL, ExAC_EAS five normal human mutation databases to retain no occurrence or less than 5% of the sites; remove the data set synonymous mutation sites, and other mutation sites reported in the literature remain.

5. Statistical analysis

The rare mutation load analysis was carried out on the filtered mutation results, and the data of 590 cases of Mykino were selected as the control sample. The OR value and 95% confidence interval were analyzed by Metabin in the R language, and the Mantel-Haenszel's method was used for statistical test, and the p value was less than 0.01 was considered significant.

6. Results

After exon sequencing, routine filtering analysis was performed. Leaving pathogenic loci, the normal frequency below 5%, and the synonymous mutation pathogenic locus reported in the literature, 59351 point mutations were obtained. In order to more accurately screen disease-related loci, further filter out SNPs predicted to be benign by SIFT, Polyphen2, MutationTaster, and GERP++; filter out Indels with Mutcount>5, MutRatio>30%, and MAF>1% in the normal database; Screening yielded 29979 point mutations.

After that, the inventors carried out the exclusion of false positive sites. First, the bam file (rmdup.sorted.bam) after de-redundant duplicates was checked and verified by IGV software, and those that did not meet the mutation conditions were regarded as false positive sites. ; At the same time, view the bam file through the samtools software. Then, using the mutated gene (a) and the mutated site (b) as the maker respectively, the rare mutation load (RVB) analysis was performed, and the number of samples with the gene mutation in the case group and the control group was counted and the OR value was calculated, OR >1,P<0.01. It was found that 48 mutation sites were statistically associated with AD.

Further, the inventors annotated the gene functions and related pathways of the 48 mutation sites screened out.

From the pathway annotations of the genes where the 48 mutation sites are located, the maps with count greater than or equal to 2 are selected for mapping, as shown in Figures 1 and 2. In the BP analysis, the genes where these sites are located are significantly enriched in redox biological processes.

In the screening results of 48 mutation sites, it was found that:

PTK6_c.205G>A_p.E69K (OR=55.65, 95%CI=2.97-1041.89) site mutation related to cell migration and differentiation. The proliferation, migration and differentiation of vascular endothelial cells can lead to morphological changes of vascular endothelial cells, which may be related to the morphological changes of vascular intima in AD.

TSHZ2_c.724C>T_p.R242C (OR=24.8, 95%CI=2.74-224.27) was associated with metal ion binding. Metal ions are closely related to vascular tension, and vascular tension may be associated with AD by affecting blood pressure.

The inventors conducted an in-depth analysis of the sequencing results by combining the annotation analysis of gene functions and related pathways, as well as the literature search data, and finally the inventors screened out two genes PTK6 and TSHZ2 that were mutated in the case group, and the mutant SNPs carried by the two genes The sites are shown in Table 1. The mutation sites of PTK6_c.205G>A_p.E69K and TSHZ2_c.724C>T_p.R242C may be the key molecules leading to AD, which can be used as new biomarkers for AD, providing an important theory for guiding its early clinical intervention and targeted therapy in accordance with.

Table 1 Aortic dissection SNP site information

Note: Gene, gene name; Chr, chromosome; Pos, SNP position; AA_change, amino acid change; ID, dbSNP annotation ID.

Example 4 Production of an early diagnosis kit for predicting aortic dissection disease

To assemble the kit for aortic dissection according to the present invention, there are three specific schemes as follows:

Kit 1: The kit includes a reagent for detecting whether the PTK6_c.205G>A_p.E69K site is mutated.

Kit 2: The kit includes a reagent for detecting whether the TSHZ2_c.724C>T_p.R242C site is mutated.

Kit 3: The kit includes reagents for detecting whether the PTK6_c.205G>A_p.E69K site and the TSHZ2_c.724C>T_p.R242C site are mutated.

The reagents include specific primers for amplifying the PTK6_c.205G>A_p.E69K site and/or the TSHZ2_c.724C>T_p.R242C site, and the primers can be designed by software, such as using Primer5, Oligo6, etc.; Said reagents can also have commonly used reagents required by corresponding PCR technology, such as: dNTPs, MgCl ₂ , double distilled water, Taq enzyme, etc., these commonly used reagents are well known to those skilled in the art, and can also have standards and controls ( Such as genotype standard and blank control, etc.). The value of this kit is that it only needs peripheral blood and no other tissue samples. It detects SNP through the most concise and specific primer pair, and then uses the SNP spectrum to assist in judging aortic dissection. It is not only stable, convenient and accurate for detection, and greatly improves the disease The sensitivity and specificity of the diagnosis, and therefore putting this kit into practice, can help guide diagnosis and more effective individualized treatment.

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (4)

1. The detection reagent for the aortic dissection susceptibility SNP sites comprises a reagent for detecting whether mutation occurs in aortic dissection susceptibility SNP sites PTK6_ c.205G > A _ p.E69K and TSHZ2_ c.724C > T _ p.R242C.

2. The detection reagent for the aortic dissection susceptibility SNP sites comprises a reagent for detecting whether mutation occurs in the aortic dissection susceptibility SNP sites PTK6_ c.205G > A _ p.E69K and TSHZ2_ c.724C > T _ p.R242C.

3. The application of the detection reagent of the SNP sites of aortic dissection susceptibility in preparing a device for predicting the risk of Stanford A-type aortic dissection diseases comprises the reagent for detecting whether the SNP sites of aortic dissection susceptibility PTK6_ c.205G > A _ p.E69K and TSHZ2_ c.724C > T _ p.R242C are mutated.

4. The use of claim 3, wherein said device is a sequencing chip.

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