Biomarker for detecting aortic dissection and application thereof

Abstract

The invention relates to the technical field of gene detection, and particularly discloses a biomarker for detecting aortic dissection and application of the biomarker. The method comprises the following steps: firstly, selecting arachidonic acid metabolism related genes according to an aortic dissection whole exon sequencing result, and searching mutation sites with relatively strong pathogenicity; secondly, the association between important gene mutation sites and arachidonic acid metabolites is clarified in combination with arachidonic acid metabonomics. In addition, through comparison with a control group, 33 gene mutations such as prostaglandin E synthetase 2 (PTGES2) and the like significantly increase the onset risk of the aortic dissection, and a theoretical basis for prognosis evaluation and genetic counseling is provided for clinical diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and particularly relates to a biomarker for detecting aortic dissection and its application. Background Art

[0002] Aortic dissection (AD) refers to a critical cardiovascular disease in which the blood in the aortic lumen enters the aortic wall through a tear in the aortic intima, causing a dissection in the middle layer of the aortic wall and extending along the longitudinal axis of the aorta. AD has an acute onset, rapid progression, and a relatively high fatality rate, bringing a heavy burden to families and society.

[0003] AD has an obvious genetic predisposition. At present, nearly 30 gene mutations have been found to increase the risk of aortic dissection onset and rupture. These genes are mainly involved in regulating the contraction of aortic smooth muscle (such as ACTA2, MYH11, MYLK, and PRKG1, etc.), the degradation of elastic fibers (such as FBN1, COL3A1, ELN, LOX, and THSD4, etc.), and the transforming growth factor-β signal transduction (such as TGFBR1, TGFBR2, SMAD3, TGFB2, and TGFB3, etc.). It is reported that these genes can only explain the causes of less than 20% of AD patients in China, which means that the pathogenic genes of most patients are still unclear. Searching for new pathogenic genes is particularly important for the early prediction and prevention of the occurrence and development of aortic dissection and its treatment. Genetic variations are closely related to the clinical phenotypes and prognoses of AD. For example, compared with AD patients without the FBN1 gene mutation, patients carrying the FBN1 gene mutation have a significantly earlier onset age, a more severe lesion degree (more often involving the ascending aorta, a larger aortic lumen diameter, and a larger range of dissection tears), and a significantly higher risk of their offspring developing dissection.

[0004] Precisely because genetic mutations play an important role in the occurrence and development of AD, current domestic and foreign guidelines in China, the United States, Europe, etc. have clearly recommended gene detection for AD patients to assist clinicians in clarifying the pathogenic causes and providing important support for subsequent diagnosis, secondary prevention, treatment, and family follow-up. Therefore, searching for new pathogenic genes and clarifying the functions of pathogenic sites are the basis for genetic testing and the development of targeted drugs, and also further deepen the understanding of the genetic mechanism of AD.

[0005] Arachidonic acid (AA) is an essential polyunsaturated fatty acid in the human body, accounting for about half in cells. The arachidonic acid metabolic pathway involves cyclooxygenase, lipoxygenase, and cytochrome P450 pathways, and their metabolites are widely involved in cardiovascular system diseases. Disorders of arachidonic acid metabolites and gene mutations of metabolic enzymes are important risk factors for diseases such as hypertension, myocardial infarction, and pulmonary hypertension, and drug treatments targeting metabolic enzymes have also been widely used in clinical practice. However, the relationship between AA metabolic gene mutations and AA metabolites and AD has not been studied and clarified. Summary of the Invention

[0006] To solve the defects and deficiencies of the prior art, the purpose of the present invention is to provide a biomarker for detecting aortic dissection and its application. The present invention relates to the discovery of arachidonic acid metabolic gene mutations and metabolite changes related to aortic dissection through whole-exome sequencing and their applications. Specifically, first, genes related to arachidonic acid metabolism were selected based on the results of whole-exome sequencing of aortic dissection to find mutation sites with stronger pathogenicity; secondly, the association between important gene mutation sites and arachidonic acid metabolites was clarified by combining arachidonic acid metabolomics. In addition, by comparing with the control group population, it was found that mutations in the prostaglandin E synthase 2 (PTGES2) gene significantly increased the risk of aortic dissection, providing a theoretical basis for prognosis assessment and genetic counseling in clinical diagnosis.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions to be realized:

[0008] In the first aspect, the present invention provides a biomarker for detecting aortic dissection, and the biomarker includes a mutant PTGES2 gene or a mutant PTGES2 protein;

[0009] Among them, the mutant PTGES2 gene has at least one of the following mutation sites relative to the coding sequence of the wild-type PTGES2 gene: c.279+2T>C, c.686+1G>A; the coding sequence of the wild-type PTGES2 gene is as shown in SEQ ID NO.1;

[0010] Among them, the mutant PTGES2 protein has at least one of the following mutation sites relative to the wild-type PTGES2 protein: p.W12X, p.T32Rfs*71, p.G68_G71del, p.L72_Y73insALGL, p.P190S, p.G199V, p.N249S, p.R252C, p.G271R, p.R304C, p.D352E; the amino acid sequence of the wild-type PTGES2 protein is as shown in SEQ ID NO.2.

[0011] As a further optimized solution of the present invention, the biomarker further includes one or more genes related to the pathogenesis of aortic dissection as follows: PLA1A, PLA2G1B, PLA2G4A, PLA2G4B, PLA2G4C, PLA2G4F, PLA2G6, PLA2G12A, CYP1A1, CYP1A2, CYP1B1, CYP2J2, CYP2S1, EPHX1, EPHX2, ALOX5, ALOX5AP, ALOX12, ALOX15, ALOXE3, PTGS1, PTGS2, PTGDS, PTGES, PTGES3, PTGIS, TBXAS1, PTGER1, PTGER2, PTGER3, PTGER4, PTGDR.

[0012] In a second aspect, the present invention provides the use of a reagent for detecting a biomarker of aortic dissection in the preparation of a kit for evaluating the risk of a patient suffering from aortic dissection, wherein the biomarker is the biomarker for detecting aortic dissection as described above.

[0013] As a further optimized solution of the present invention, the reagent contains a primer pair for detecting the mutant PTGES2 gene.

[0014] As a further optimized solution of the present invention, the primer pair includes at least one of the following groups: SEQ ID NO.3 and SEQ ID NO.4; SEQ ID NO.5 and SEQ ID NO.6; SEQ ID NO.7 and SEQ ID NO.8; SEQ ID NO.9 and SEQ ID NO.10.

[0015] As a further optimized solution of the present invention, the biomarker further includes at least one arachidonic acid metabolite among PGE2, PGD2, PGF2α, HTETs, and LTB4.

[0016] As a further optimized solution of the present invention, the kit further includes a liquid chromatography-tandem mass spectrometry reagent for detecting the concentration of arachidonic acid metabolites in plasma.

[0017] As a further optimized solution of the present invention, the reagent contains a specific antibody for detecting the mutant PTGES2 protein.

[0018] In a third aspect, the present invention provides a kit, characterized in that the kit contains a reagent for detecting the biomarker as described above.

[0019] The present invention conducts research on the biomarker of aortic dissection and provides an important basis for the diagnosis and treatment of aortic dissection through various experiments. The specific beneficial effects are as follows:

[0020] 1. The associations of 33 genes related to arachidonic acid metabolism with aortic dissection (AD) were clarified. In particular, the PTGES2 gene is a novel AD genetic susceptibility gene. Among 2,006 patients, 15.6% of the patients carried at least one mutation related to the AA gene. Among them, the PTGES2 gene had the highest mutation frequency, and its genetic mutations were significantly enriched in AD patients, providing a new direction for in-depth understanding of the genetic mechanism of AD.

[0021] 2. Functional experiments on PTGES2 gene variations were conducted. It was found that loss-of-function mutations caused premature termination of mPGES-2 protein translation, and missense mutations reduced its expression level. It was confirmed that the c.686+1G>A variation disrupted the classical alternative splicing site to generate a new transcript, determining the pathogenicity of PTGES2 genetic variation sites and contributing to the precise analysis of the molecular mechanism of AD onset.

[0022] 3. Mass spectrometry technology was used to detect arachidonic acid metabolites in the plasma of AD patients and the control group. It was found that various prostaglandins and pro-inflammatory metabolites in the plasma of AD patients were significantly increased. In patients carrying PTGES2 gene mutations, the increase in pro-inflammatory metabolites was more obvious. For example, the concentrations of PGE2, PGD2, PGF2α, and various HETEs and LTB4 were all significantly increased, providing new biological indicators for the diagnosis and disease condition assessment of the disease.

[0023] 4. Reagents for detecting 33 genes related to arachidonic acid metabolism, especially PTGES2 gene mutations, as well as kits and applications for detecting arachidonic acid substances after gene mutations were provided. It can be used to prepare kits for evaluating the risk of patients with aortic dissection, which is of great significance for early prediction and prevention of the disease, and provides a powerful tool for clinical diagnosis, prognosis assessment, and genetic counseling. Description of the Drawings

[0024] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, form a part of this application, and do not limit the embodiments of the present invention. In the drawings:

[0025] Figure 1 It is a distribution map of rare harmful variations of genes in the arachidonic acid metabolism pathway of aortic dissection patients.

[0026] Figure 2 It is the effect of PTGES2 point mutations on mPGES-2 protein expression; A: Loss-of-function mutation, B: Non-frameshift mutation and missense mutation, WT: Wild type.

[0027] Figure 3 It is the c.686+1G>A variation generating a new transcript; A: MiniGene experimental pattern diagram and gel diagram, B: First-generation sequencing results.

[0028] Figure 4 The decrease in the percentage of alternative splicing is caused by the c.279+2T>C alternative splicing variant; A: Schematic diagram and gel image of the MiniGene experiment, B: First-generation sequencing map.

[0029] Figure 5 The heat map shows the changes in the content of arachidonic acid metabolites caused by different gene mutations.

[0030] Figure 6 Knockout of the PTGES2 gene exacerbates the incidence of aortic dissection in male mice; A: Representative gross images after 4 weeks of BAPN administration in drinking water, B: Mouse survival curve, C: Statistical chart of the maximum aortic diameter of mice, * represents p<0.05, **** represents p<0.0001. Detailed implementation manners

[0031] The present invention will be further described in detail below with reference to the accompanying drawings, examples and experimental examples. Of course, the protection scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit of the present invention. The present invention generally and / or specifically describes the materials and test methods used in the experiments. Although many materials and operation methods used to achieve the purpose of the present invention are well known in the art, the present invention still describes them in as much detail as possible herein. The following examples further illustrate the present invention, rather than limiting the present invention. Any equivalent transformation that is merely formal rather than substantial based on the concept of the present invention should be regarded as falling within the scope of the technical solution of the present invention.

[0032] For those not specifying specific techniques or conditions in the examples, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular channels.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0034] I. At least one of 15.6% of Chinese patients with aortic dissection carries a rare harmful variant related to arachidonic acid metabolism genes

[0035] 1. Study population

[0036] This study relied on Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology and the Taizhou Population Health Follow-up Survey Cohort. A total of 2,006 patients with sporadic thoracic aortic dissection were included by the researchers. All patients were clearly diagnosed with aortic dissection by Doppler ultrasound, computed tomography, magnetic resonance imaging, angiography or surgery. Whole-exome sequencing was performed on all patients to detect point mutations in the PTGES2 gene.

[0037] 2. Research methods

[0038] For all patients, the Agilent SureSelect Human All Exon V6 microarray capture system was used to enrich the genomic exon regions. The constructed libraries were quality-checked using an ABI Q3 qPCR instrument, and whole-exome sequencing was performed using the Illumina NovaSeq platform. Variation sites on the exons of 33 AA-related genes were evaluated (Table 1), mainly including nonsense mutations, frameshift mutations, classical splice site mutations, and rare mutations with a predicted harmful mutation frequency less than 0.005 by bioinformatics software.

[0039] 3. Results

[0040] According to the screening process, a total of 179 rare harmful mutations were identified. Among them, 48 (26.8%) were loss-of-function mutations, 119 (66.5%) were missense mutations, and 12 (6.7%) were in-frame mutations. Among the loss-of-function mutations, 17 were frameshift insertion / deletion mutations, 23 were nonsense mutations, and 8 were alternative splicing variants. All these mutations were distributed in 312 patients (312 / 2,006, 15.6%). The gene with the highest mutation frequency was PTGES2, accounting for 14% of the total number of mutated patients (43 / 312, Figure 1 ), followed by CYP1A1 (35 / 312, 11%), CYP2J2 (34 / 312, 11%), PTGS1 (31 / 312, 10%), and PTGIS (30 / 312, 10%). These mutations were all significantly associated with AD and were its risk-causing genes.

[0041] II. Rare harmful mutations in PTGES2 increase the risk of aortic dissection

[0042] 1. Research population

[0043] Patient group: 2006 AD patients were divided into a discovery cohort and a validation cohort. The discovery cohort consisted of 1171 patients diagnosed with AD before July 2020 at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The validation cohort included 835 patients diagnosed with AD from AD dissections at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from July 2020 to August 2023 and patients diagnosed with AD from the Taizhou Population Health Follow-up Survey Cohort of Fudan University.

[0044] Control group: Public genetic databases of The China Metabolic Analytics Project (ChinaMap) and The Westlake BioBank for Chinese (WBBC) were used as controls. The China Metabolic Analytics Project performed whole-genome sequencing on 10,588 Chinese individuals from different regions and ethnic groups in China. The Westlake BioBank for Chinese published research results on genomic genetic mutations involving 10,376 samples from 29 administrative regions in China through whole-genome sequencing and gene chip genotyping technologies, and currently, the genetic information of 4,480 samples is publicly available.

[0045] 2. Research methods

[0046] The variant sites on the exon of PTGES2 were evaluated, mainly including nonsense mutations, frameshift mutations, classic splice site mutations, and rare mutations with a predicted harmful mutation frequency less than 0.005 by bioinformatics software. The chi-square test was used to compare the difference in mutation frequencies between patients and the control group, and the relative risk, 95% confidence interval, and p-value of the risk alleles were calculated.

[0047] 3. Results

[0048] Among all 2,006 patients, we identified 13 PTGES2 gene mutation sites in 43 patients, including 4 loss-of-function mutations and 9 point mutations, with a genetic interpretability of 2.14%. Table 2 shows the specific variant information of PTGES2 gene mutations. Further, by comparing with the control population, we found that in the discovery cohort, rare harmful variants of PTGES2 increased the risk of aortic dissection (ChinaMap: OR 2.16, 95% CI 1.35–3.36, p<0.001; WBBC: OR 2.23, 95% CI 1.32–3.72, p<0.001; Combined: OR 2.18, 95% CI 1.38–3.34, p<0.001). This result was verified in the second population (ChinaMap: OR 1.97, 95% CI 1.1–1.32, p = 0.0086; WBBC: OR 2.04, 95% CI 1.09–3.65, p = 0.01; Combined: OR 1.99, 95% CI1.13–3.31, p = 0.0065). When the two populations were combined, the significance was even more remarkable (ChinaMap: OR 2.09, 95% CI 1.43–3, p<0.001; WBBC: OR 2.16, 95% CI 1.36–3.37, p<0.001; Combined: OR2.11, 95% CI 1.46–2.98, p<0.001).

[0049] III. Cytological experiments verify that PTGES2 variants affect the mPGES-2 protein level

[0050] To further explore the effect of variant sites on the PTGES2 gene, we first constructed overexpression plasmids of wild-type and mutant PTGES2, and explored the effect of point mutations on the expression of mPGES-2 protein through the expression level of the tag V5.

[0051] 1. Construction of mutant plasmids

[0052] The cDNA sequence (NM_025072) of the human PTGES2 gene was artificially synthesized and cloned into the pcDNA3.1-His-V5 vector, and 11 mutant plasmids were constructed through a site-directed mutagenesis kit (Novizan Mut II Fast Mutagenesis Kit V2 kit) (Table 3), and the wild-type and mutant plasmids were sequenced.

[0053] 2. Transfection

[0054] The plasmid was transfected into human embryonic kidney cells (293T) using Lipofectamine 2000 transfection reagent (Invitrogen). 48 hours after transfection, the expression level of mPGES-2 protein was detected using Western blotting.

[0055] 3. Results

[0056] Figure 2 A shows the positions of the p.W12X truncating mutation and the p.T32Rfs*71 frameshift mutation in the PTGES2 gene. Western blotting results indicated that these two mutations caused premature termination of mPGES-2 translation, resulting in the inability to express the full-length mPGES-2 protein. Figure 2 B shows the positions of the remaining missense mutations in the PTGES2 gene. Western blotting results indicated that except for the p.G68_G71del and p.L72_Y73insALGL mutations that did not cause changes in the expression level of mPGES-2 protein, the remaining point mutations significantly reduced the expression of mPGES-2 protein.

[0057] IV. MiniGene experiment to verify the functions of the c.279+2T>C and c.686+1G>A point mutations

[0058] 1. Methods

[0059] The vector used in the MiniGene experiment in this study was the pMini-CopGFP empty vector. The vector was linearized using the restriction endonucleases BamHⅠ and XhoⅠ, and the linearized vector and the inserted fragment were recombined by homologous recombination to construct the specific Minigene splicing site mutant plasmids for this study (Tables 3 and 4).

[0060] 2. Results

[0061] Sequencing results indicated that the c.686+1G>A variant disrupted the classical alternative splicing site, causing exon 4 of transcript NM_025072 to skip, thereby generating a new transcript ( Figure 3 A-3B). Although the c.279+2T>C variant did not generate a new transcript, compared with the wild type, the expression level of its theoretical transcript was significantly reduced, manifested as a decrease in the band brightness after mutation, suggesting that this variant led to a decrease in the percentage of alternative splicing ( Figure 4 A-4B).

[0062] V. Effects of gene mutations on the contents of arachidonic acid metabolites

[0063] 1. Study population

[0064] 255 AD patients from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

[0065] 2. Detection

[0066] A method of liquid chromatography - tandem mass spectrometry was used to detect a variety of metabolites including arachidonic acid, prostaglandins, hydroxyeicosatetraenoic acid (HEHE), dihydroxyeicosatrienoic acid, and epoxyeicosatrienoic acid. For normally distributed measurement data, we used the mean ± standard deviation for description, and the independent samples t - test was used for comparison between two groups; skewed measurement data were described using the median (interquartile range), and the Mann–Whitney U - test was used for comparison between two groups.

[0067] 3. Results

[0068] We compared the changes in arachidonic acid metabolites in the plasma of patients carrying arachidonic acid - related gene mutations with those of patients not carrying arachidonic acid - related gene mutations, and found that in patients carrying the PTGES2 gene mutation, pro - inflammatory metabolites increased significantly. Among prostaglandin metabolites, after carrying the PTGES2 gene mutation, the concentrations of PGE 2 , PGD 2 and PGF 2α all increased significantly (p < 0.05). Among HETEs metabolites, the concentrations of all HTETs in the group carrying the PTGES2 gene mutation increased, specifically 5 - HETE, 8 - HETE, 9 - HETE, and 11 - HETE. In addition, the downstream product of 5 - HETE, leukotriene LTB4, also increased significantly in the group of patients carrying the PTGES2 gene. In addition, in patients carrying the PTGIS gene mutation, the content of PGD2 increased significantly, and 8 - HETE, 9 - HETE, 11 - HETE, and LTB4 also increased. While in patients carrying the CYP2J2 gene mutation, the content of EET decreased significantly, specifically the contents of 5,6 - EET and 11,12 - EET decreased ( Figure 5 ).

[0069] VI. Global knockout of PTGES2 promotes the incidence and mortality of BAPN - induced thoracic aortic dissection aneurysm

[0070] The above - mentioned functional studies showed that the PTGES2 mutation we found could cause a decrease in the mPGES - 2 protein level. Therefore, we used the CRISPR / Cas9 technology to construct PTGES2 global knockout mice. Further, the effect of PTGES2 knockout (PTGES2 - KO) on the incidence of aortic dissection was evaluated in a mouse model.

[0071] 1. Model construction

[0072] In this experiment, 0.5% β-aminopropionitrile (BAPN) was used to induce aortic dissection in 4-week-old mice. 0.5 g of BAPN was added to 100 mL of drinking water, and the water was changed every 3 days for 28 days. Since BAPN is easily decomposed by light, black plastic bags were wrapped around the water bottles to ensure light avoidance and thus guarantee the stability of the drug.

[0073] 2. Animal grouping

[0074] To explore the effect of global PTGES2 knockout on aortic dissection, we set up the following groups: ① wild-type (WT) + saline group; ② WT + BAPN group; ③ PTGES2-KO + saline group; ④ PTGES2-KO + BAPN group. During this period, the mice were observed daily for any deaths. If a death occurred, the mouse was immediately dissected to determine the cause of death.

[0075] 3. Experimental results

[0076] We fed 4-week-old homozygous PTGES2-KO male mice and littermate wild-type male mice with 0.5% BAPN for 28 consecutive days to induce aortic dissection aneurysm in the mice. We found that compared with wild-type mice, PTGES2 gene knockout aggravated the incidence of BAPN-induced aortic dissection in mice. By measuring the aortic internal diameter to evaluate the aortic dilation of the two groups of mice, we found that the maximum aortic diameter of PTGES2 gene knockout mice was significantly widened ( Figure 6 ).

[0077] Table 1

[0078]

[0079]

[0080] Table 2: Specific mutation information of PTGES2 gene

[0081]

[0082]

[0083] Table 3: Point mutation primers

[0084]

[0085]

[0086] Table 4: MiniGene primers

[0087]

[0088]

[0089] A total of 2,006 patients with aortic dissection were studied in this invention. Among them, 1,171 AD patients were from the discovery cohort, and 835 patients were from the validation cohort. First, we performed whole-exome or whole-genome sequencing on all patients and analyzed gene mutations in 33 major genes involved in arachidonic acid metabolism. We found that 15.6% of the patients carried at least one related mutation in the AA gene. Among them, the mutation frequency of the PTGES2 gene was the highest (43 / 312, accounting for 13.8% of the total number of patients). Subsequently, the relatively high mutation frequencies were CYP1A1 (35 / 312, 11%), CYP2J2 (34 / 312, 11%), PTGS1 (31 / 312, 10%), and PTGIS (30 / 312, 10%). In addition, we also found that genetic mutations in the PTGES2 gene were significantly enriched in AD patients. Further, we included two large Chinese genetic data populations as controls for gene burden testing and found that rare harmful variants in the PTGES2 gene were closely related to the risk of AD onset.

[0090] Subsequently, we conducted functional experiments on the variants found above. We found that loss-of-function mutations in PTGES2 could prematurely terminate the translation of the mPGES-2 protein; missense mutations could significantly reduce the expression level of the mPGES-2 protein; and the miniGene technology confirmed that the splicing site c.686+1G>A variant disrupted the classical alternative splicing site, causing exon 4 of the transcript NM_025072 to skip, thus generating a new transcript. These functional experiments determined the pathogenicity of the PTGES2 genetic variant sites.

[0091] Subsequently, we detected the changes in 126 arachidonic acid metabolites in the plasma of AD patients and control subjects by mass spectrometry. We found that compared with the control group, the levels of various prostaglandins in the plasma of AD patients were significantly increased, including PGB2, PGE2, PGD2, and PGF2α, etc. Pro-inflammatory metabolites such as HETEs (5-HETE, 8-HETE, 9-HETE, and leukotrienes, etc.) and DHETs (5,6-DHET, 8,9-DHET, 14,15-DHET) were also significantly increased. Further, we compared the changes in arachidonic acid metabolites in the plasma of patients carrying arachidonic acid-related gene mutations with those of patients not carrying arachidonic acid-related gene mutations, and found that in patients carrying PTGES2 gene mutations, the pro-inflammatory metabolites were significantly increased. Among the prostaglandin metabolites, after carrying the PTGES2 gene mutation, the concentrations of PGE2, PGD2, and PGF2α were all significantly increased (p<0.05). Among the HETEs metabolites, all HTETs (such as 5-HETE, 8-HETE, 9-HETE, and 11-HETE) and the downstream product leukotriene LTB4 of 5-HETE in the group of patients carrying the PTGES2 gene were also significantly increased. In addition, in patients carrying the PTGIS gene mutation, PGD2, LTB4, 8-HETE, 9-HETE, and 11-HETE were also increased. In patients carrying the CYP2J2 gene mutation, the content of EET was significantly decreased, specifically manifested as the decrease in the content of 5,6-EET and 11,12-EET.

[0092] Finally, we constructed PTGES2 knockout mice to explore the effect of PTGES2 knockout on the pathogenesis of aortic dissection at the animal level. We found that compared with wild-type mice, PTGES2 gene knockout aggravated the pathogenesis of BAPN-induced aortic dissection in mice. By measuring the aortic diameter to evaluate the aortic dilation of the two groups of mice, we found that the maximum aortic diameter of PTGES2 gene knockout mice was significantly widened.

[0093] In summary, the present invention identified 33 genes related to arachidonic acid metabolism, especially the PTGES2 gene is a novel AD genetic susceptibility gene, and provided reagents for detecting 33 genes related to arachidonic acid metabolism, especially kits for detecting PTGES2 gene mutations and the application of detecting arachidonic acid substances after gene mutation.

[0094] The above is only the preferred embodiment of the present invention. It should be noted that the above preferred embodiment should not be construed as a limitation of the present invention. The protection scope of the present invention should be determined by the scope defined in the claims. For those of ordinary skill in the art in this technical field, without departing from the spirit and scope of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A biomarker for detecting aortic dissection, characterized in that: The biomarkers include mutant PTGES2 gene or mutant PTGES2 protein; The mutant PTGES2 gene has at least one of the following mutation sites relative to the coding sequence of the wild-type PTGES2 gene: c.279+2T>C, c.686+1G>A; the coding sequence of the wild-type PTGES2 gene is shown in SEQ ID NO.1; Among them, the mutant PTGES2 protein has at least one of the following mutation sites relative to the wild-type PTGES2 protein: p.W12X, p.T32Rfs*71, p.G68_G71del, p.L72_Y73insALGL, p.P190S, p.G199V, p.N249S, p.R252C, p.G271R, p.R304C, p.D352E; the amino acid sequence of the wild-type PTGES2 protein is shown in SEQ ID NO.

2.

2. The biomarker for detecting aortic dissection according to claim 1, characterized in that: The biomarkers also include one or more of the following aortic dissection pathogenicity-related genes: PLA1A, PLA2G1B, PLA2G4A, PLA2G4B, PLA2G4C, PLA2G4F, PLA2G6, PLA2G12A, CYP1A1, CYP1A2, CYP1B1, CYP2J2, CYP2S1, EPHX1, EPHX2, ALOX5, ALOX5AP, ALOX12, ALOX15, ALOXE3, PTGS1, PTGS2, PTGDS, PTGES, PTGES3, PTGIS, TBXAS1, PTGER1, PTGER2, PTGER3, PTGER4, and PTGDR.

3. Use of a reagent for detecting a biomarker of aortic dissection in the preparation of a kit for assessing the risk of aortic dissection in a patient, characterized in that: The biomarker is the biomarker for detecting aortic dissection according to claim 1.

4. The use according to claim 3, characterized in that: The reagent contains a primer pair for detecting the mutant PTGES2 gene.

5. The use according to claim 4, characterized in that: The primer pair includes at least one of the following: SEQ ID NO.3 and SEQ ID NO.4; SEQ ID NO.5 and SEQ ID NO.6; SEQ ID NO.7 and SEQ ID NO.8; SEQ ID NO.9 and SEQ ID NO.

10.

6. The use according to claim 3, characterized in that: The biomarkers also include PGE2, PGD2, PGF 2α , HTETs, and at least one arachidonic acid metabolite among LTB4.

7. The use according to claim 6, characterized in that: The kit also includes a liquid chromatography-tandem mass spectrometry reagent for detecting the concentration of arachidonic acid metabolites in plasma.

8. The use according to claim 3, characterized in that: The reagent contains a specific antibody for detecting mutant PTGES2 protein.

9. A kit, characterized in that: The kit comprises reagents for detecting the biomarkers described in claim 1.

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