N-carbohydrate chain marker for diagnosing type 2 hereditary angioedema, kit and application of N-carbohydrate chain marker

Abstract

The invention relates to the technical field of medical detection, in particular to an N-carbohydrate chain marker for diagnosing type 2 hereditary angioedema, a kit and application of the N-carbohydrate chain marker. The content of the N-carbohydrate chain marker provided by the invention is obviously changed in the body of a patient with type-2 hereditary angioedema, and verification shows that the markers all show good diagnostic ability of type-2 hereditary angioedema, can effectively distinguish the patient with type-2 hereditary angioedema from a healthy human body and a non-HAE edema patient, and can be used for detecting the type-2 hereditary angioedema. The molecular marker can be used as a marker for diagnosing type 2 hereditary angioedema, and has high specificity, sensitivity and accuracy. The N-carbohydrate chain marker has the advantages of convenience in detection, short required time and the like when being used for diagnosing the type 2 hereditary angioedema, and has a better application prospect in clinical diagnosis of the type 2 hereditary angioedema.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to an N-sugar chain marker, a kit and an application thereof for diagnosing hereditary angioedema type 2. Background Art

[0002] Hereditary angioedema (hAE) is a rare, life-threatening genetic disease with an incidence of approximately 1 / 50,000. The clinical manifestations are highly heterogeneous and are characterized by acute, recurrent subcutaneous and / or submucosal tissue edema, which can affect the face, limbs, upper respiratory tract, and gastrointestinal tract. Classic HAE includes HAE type 1 (HAE-1) and HAE type 2 (HAE-2), of which the incidence of HAE-2 is much lower than that of HAE-1. At present, the diagnosis of HAE internationally requires a combination of family history, clinical manifestations, and laboratory tests. However, due to the rarity of HAE, clinical heterogeneity, and defects in existing diagnostic markers, patients are often misdiagnosed and mistreated, and the diagnosis of HAE is delayed.

[0003] The laboratory examination items for HAE are C4 concentration, C1 esterase inhibitor (C1-INH) concentration and function. Since C4 can be reduced in many diseases, the specificity of C4 detection is poor and can only be used as an auxiliary reference indicator. The concentration and function of C1-INH are specific indicators for the diagnosis of HAE-2. Since there is no significant difference in C1-INH concentration between HAE-2 patients and non-HAE edema patients, the functional detection of C1-INH is a necessary way to diagnose HAE-2. However, C1-INH is easy to degrade, and its functional detection has extremely high requirements for sample collection, preparation, transportation and storage in the early stage, making it difficult to popularize in clinical practice. Chinese patent application CN113311056A discloses markers for the diagnosis of hereditary angioedema, but it is for HAE-1 type rather than HAE-2 type. Therefore, there is an urgent need for highly specific and convenient HAE-2 diagnostic markers in clinical practice.

[0004] Protein glycosylation is one of the most common and complex post-translational modifications. The glycans on proteins mainly play a role by participating in a variety of important biological processes such as cell adhesion, protein folding, molecular transport and clearance, receptor activation and signal transduction. Therefore, protein glycosylation can significantly affect the structure and function of proteins. Protein glycosylation can be divided into N-glycosylation, O-glycosylation, C-mannosylation and glycosylphosphatidylinositol anchoring, of which more than half of glycoproteins are N-glycosylated. In recent years, the development of glycomics technology has enabled a more comprehensive understanding of the biological significance of protein glycosylation in physiological and disease states. Glycomics has a more prominent diagnostic and predictive ability in many aspects such as disease diagnosis, prediction of progression and prognosis. Studies have found that changes in the N-glycan spectrum in plasma are associated with the occurrence and development of a variety of diseases, such as lung cancer, colorectal cancer and other cancers, Alzheimer's disease, type 2 diabetes cardiovascular disease, etc. Summary of the invention

[0005] The present invention provides an N-sugar chain marker for diagnosing type 2 hereditary angioedema, a kit and applications thereof.

[0006] The present invention develops sugar chain markers with diagnostic significance for hereditary angioedema type 2, detects and analyzes the plasma full glycome profiles of clinical hereditary angioedema type 2 patients, healthy subjects, and patients with mast cell-mediated edema, analyzes the specific N-sugar chain characteristics of hereditary angioedema type 2 patients, screens N-sugar chain markers related to hereditary angioedema type 2, and determines potential markers for the diagnosis of hereditary angioedema type 2 by methods such as ROC curve analysis.

[0007] Specifically, the present invention provides the technical solution described below.

[0008] In a first aspect, the present invention provides a marker for diagnosing hereditary angioedema type 2, wherein the marker comprises a combination of one or more of the following sugar chain markers: H9N2, H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, H3N4F1, H4N4F1, MM, CA2, CA3, CF0, CG, CS; Alternatively, the markers include one or more selected from H4N5F1E1, H5N4F1L2, CF and one or more selected from H9N2, H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, H3N4F1, H4N4F1, MM, CA2, CA3, CF0, CG, CS.

[0009] Compared with healthy subjects and patients with mast cell-mediated edema, the levels of the above-mentioned N-glycan markers in the plasma of patients with hereditary angioedema type 2 were significantly changed, and the AUC values ​​in the diagnosis of hereditary angioedema type 2 were all above 0.75. They can more accurately diagnose hereditary angioedema type 2 and differentiate hereditary angioedema type 2 from non-HAE edema, with high specificity and sensitivity.

[0010] In some embodiments of the present invention, the marker comprises one selected from H9N2, H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, H3N4F1, H4N4F1, MM, CA2, CA3, CF0, CG, and CS.

[0011] Since the AUC value of any one of H9N2, H5N4E1, H4N5F1E1, H5N4F1L2, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, H3N4F1, H4N4F1, MM, CA2, CA3, CF0, CG, CS, and CF in diagnosing hereditary angioedema type 2 is above 0.75, these markers can be used alone to diagnose hereditary angioedema type 2, and any 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of them can also be used in combination. When used in combination, the AUC value for the diagnosis of hereditary angioedema type 2 must also be above 0.75, or even higher than the AUC value of each individual marker in the combination. Therefore, a combination of at least two of the above-mentioned sugar chain markers can also be used to diagnose hereditary angioedema type 2 with high accuracy.

[0012] In some embodiments of the present invention, the markers include at least two selected from H9N2, H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, H3N4F1, H4N4F1, MM, CA2, CA3, CF0, CG, and CS; or, include one or more selected from H4N5F1E1, H5N4F1L2, and one or more selected from H9N2, H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, H3N4F1, H4N4F1, MM, CA2, CA3, CF0, CG, and CS.

[0013] Preferably, in patients with hereditary angioedema type 2, the expression levels of H9N2, H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, MM, CA3, CF0, CS, and CG are significantly decreased compared with those in healthy humans and patients with mast cell-mediated edema; the expression levels of H4N5F1E1, H5N4F1L2, H3N4F1, H4N4F1, CA2, and CF are significantly increased compared with those in healthy humans and patients with mast cell-mediated edema.

[0014] In addition, the present invention also found that in the plasma N-glycan characteristics of patients with hereditary angioedema type 2, the combination of fucosylated (F), alpha2,6-linked sialic acid (E) or alpha2,3-linked sialic acid (L) can also be used to diagnose hereditary angioedema type 2, and its AUC value can reach more than 0.93, with high specificity and sensitivity. Therefore, the above marker combination can also be used to diagnose hereditary angioedema type 2.

[0015] In some embodiments of the present invention, the markers include H4N5F1E1, H5N4F1L2, H3N4F1 and H4N4F1; or, include H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2 and H4N5F1E1; or, include H6N5E2L1, H6N5E1L1, H6N5E1L2 and H5N4F1L2.

[0016] Preferably, in patients with hereditary angioedema type 2, the expression levels of H4N5F1E1, H5N4F1L2, H3N4F1 and H4N4F1 (i.e., F panel), H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2 and H4N5F1E1 (i.e., E panel), and H6N5E2L1, H6N5E1L1, H6N5E1L2 and H5N4F1L2 (i.e., L panel) are significantly increased compared with those in healthy subjects and patients with mast cell-mediated edema.

[0017] The naming method of the sugar chain markers described in the present invention refers to the following document: Zhang Z, Westhrin M, Bondt A, et al. Serum protein N-glycosylation changes in multiple myeloma[J]. Biochimica et Biophysica Acta (BBA)-General Subjects, 2019. In the present invention, the N-sugar chain markers include sugar chain structures that can be directly detected by mass spectrometry, and the names and molecular weights of these sugar chain structures are shown in Table 1. In addition to the sugar chain structures directly detected by mass spectrometry, derived glycan traits are calculated by Rstudio based on the directly detected sugar chain structures according to their structural characteristics and biological relevance. Derived glycan features were calculated based on the directly detected glycan structural features and the biosynthetic pathways they reflected: glycan type (high mannose (M), complex (C), and hybrid (Hy) glycans), number of antennae / branches (A), and other features such as bisecting glycans (B), galactosylation (G), fucosylation (F), and linkage-specific sialylation (S). The first set of derived glycan features divided all directly detected glycans into high mannose (M), complex (C), and hybrid (Hy) glycans. Complex (C) glycans were then further subdivided based on the number of antennae / branches, the presence and number of fucosylation, and the content of galactosylation and sialylation in the glycoform. The derived glycan features indicate that glycan changes occur together in a group of structurally related glycans. The subject of the calculation is indicated by the last letter, for example, sialylation (S), and the letter before S indicates the range of the calculation, such as fucosylated biantennary sugar chains (A2F). Therefore, A2FS can be translated as the sialylation level in fucosylated biantennary sugar chains.

[0018] Derived glycan features include: the number of antennae of complex-type glycans (CA), the level of fucosylation (F), the level of bisection (B), the level of terminal galactosylation (G), the level of sialylation (S), etc. The specific structure and calculation formula of each N-glycan marker refer to Zhang Z, Westhrin M, Bondt A, et al. Serumprotein N-glycosylation changes in multiple myeloma[J]. Biochimica etBiophysica Acta (BBA)-General Subjects, 2019.

[0019] Table 1

[0020] In a second aspect, the present invention provides use of the above-mentioned marker for diagnosing hereditary angioedema type 2 or a detection reagent thereof in the preparation of a product for diagnosing hereditary angioedema type 2.

[0021] Preferably, the product is a medicine or a kit.

[0022] In a third aspect, the present invention provides use of the above-mentioned markers for diagnosing hereditary angioedema type 2 as drug targets for preventing or treating hereditary angioedema type 2.

[0023] In a fourth aspect, the present invention provides a product for the diagnosis of hereditary angioedema type 2, wherein the product comprises the above-mentioned marker for the diagnosis of hereditary angioedema type 2 or a detection reagent thereof.

[0024] Preferably, the product is a medicine or a kit.

[0025] In the present invention, the detection reagent of the marker can be any reagent used to detect the marker of the present invention, for example: characteristic sugar chain probe, mass spectrometry detection reagent, lectin chip, etc.

[0026] In some embodiments of the present invention, the detection reagent is a characteristic sugar chain probe that specifically recognizes the sugar chain marker.

[0027] In some embodiments of the present invention, the detection reagent is a mass spectrometry detection reagent for detecting the sugar chain marker.

[0028] In a fifth aspect, the present invention provides a device for diagnosing hereditary angioedema type 2, the device comprising: A detection module, used to detect the content of the above-mentioned markers for diagnosing hereditary angioedema type 2 in the sample to be tested; An input module is used to obtain the detection result of the detection module; A judgment module, used to judge whether it is hereditary angioedema type 2 according to the test result obtained by the input module; The output module is used to output the judgment result.

[0029] Preferably, the judgment standard of the judgment module is: in patients with hereditary angioedema type 2, the expression levels of H9N2, H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, MM, CA3, CF0, CS, and CG are significantly decreased compared with those in healthy humans and patients with mast cell-mediated edema; the expression levels of H4N5F1E1, H5N4F1L2, H3N4F1, H4N4F1, CA2, and CF are significantly increased compared with those in healthy humans and patients with mast cell-mediated edema.

[0030] Preferably, the sample to be tested is plasma.

[0031] In a sixth aspect, the present invention provides a method for diagnosing hereditary angioedema type 2, the method comprising: detecting the content of the above-mentioned markers for diagnosing hereditary angioedema type 2 in the subject's plasma, and diagnosing whether it is hereditary angioedema type 2 based on the content of the markers.

[0032] Preferably, in patients with hereditary angioedema type 2, the expression levels of H9N2, H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, MM, CA3, CF0, CS, and CG are significantly decreased compared with those in healthy humans and patients with mast cell-mediated edema; the expression levels of H4N5F1E1, H5N4F1L2, H3N4F1, H4N4F1, CA2, and CF are significantly increased compared with those in healthy humans and patients with mast cell-mediated edema.

[0033] The beneficial effects of the present invention include at least: the content of the N-sugar chain marker provided by the present invention changes significantly in patients with hereditary angioedema type 2, and the ROC curve verifies that both show good ability to distinguish hereditary angioedema type 2, and can effectively distinguish hereditary angioedema type 2 patients from healthy people and non-HAE edema patients, and can be used as a marker for the diagnosis of hereditary angioedema type 2, with high specificity, sensitivity and accuracy. The use of the N-sugar chain marker of the present invention for the diagnosis of hereditary angioedema type 2 also has the advantages of convenient detection and short time required, and has a good application prospect in the clinical diagnosis of hereditary angioedema type 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 3D PCA (A) and 3D PLS-DA (B) analysis results of the total plasma N-glycome in patients with hereditary angioedema type 2 and mast cell-mediated edema and healthy controls in Example 1 of the present invention.

[0036] Figure 2 It is a box plot of the changes in glycan traits between patients with hereditary angioedema type 2 (HAE-2), patients with mast cell-mediated edema (MC-AE) and healthy controls (HC) in Example 1 of the present invention.

[0037] Figure 3 : This is a ROC curve diagram of the diagnostic N-glycan marker for hereditary angioedema type 2 in Example 1 of the present invention (before covariate adjustment).

[0038] Figure 4 : This is a ROC curve diagram of the diagnostic N-glycan marker for hereditary angioedema type 2 in Example 1 of the present invention (after covariate adjustment).

[0039] Figure 5 : This is the ROC curve of the diagnostic N-glycan F, E, and L combination for hereditary angioedema type 2 in Example 1 of the present invention, wherein A, B, and C are ROC curves without covariate adjustment, and D, E, and F are ROC curves after covariate adjustment.

[0040] Figure 6It is a box plot of the changes in derived glycan traits between patients with hereditary angioedema type 2 (HAE-2), patients with mast cell-mediated edema (MC-AE) and healthy controls (HC) in Example 1 of the present invention.

[0041] Figure 7 : is the ROC curve diagram of the diagnostic N-glycan marker (derived) for hereditary angioedema type 2 in Example 1 of the present invention (after covariate adjustment).

[0042] Figure 3-Figure 5 as well as Figure 7 In the figure, the horizontal axis is the false positive rate (FPR), which is 1-specificity; the vertical axis is the true positive rate (TPR), which is sensitivity. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The "glycome" mentioned in the present invention refers to all sugar chains expressed in a sample (such as body fluids, cells, tissues) or all sugar chains on a certain type of glycoprotein.

[0045] The sample of the present invention is selected from: body fluid samples, such as blood, serum, plasma, urine, saliva, cerebrospinal fluid, lymph, spinal fluid, ascites, amniotic fluid; cell samples, such as cell samples separated from tissues, cell samples cultured in vitro; tissue samples, which can be in the form of fresh tissue samples, fixed tissue samples, etc. In specific detection practice, plasma samples are preferred.

[0046] The sugar chains described in the present invention may be free sugar chains or sugar chains released from sugar complexes.

[0047] Free sugar chains can be obtained by techniques known in the art, including but not limited to: enzymatic methods, such as using glycosidase, preferably glycosidase PNGase F; chemical methods, such as using β-elimination reaction, glycoprotein hydrazinolysis reagent; and a combination of enzymatic and chemical methods can also be used to release sugar chains.

[0048] The derivatization methods of the present invention include but are not limited to: methyl amination, esterification, methylation, reductive amination, acetylation, etc. The type of derivatization can be selected according to the needs, and esterification is preferred.

[0049] In the present invention, after the sugar chains are released from the body fluid protein, the N-sugar chains can be purified and / or enriched using techniques known in the art. The purification and enrichment methods include, but are not limited to, centrifugation, filtration, extraction, adsorption, capillary electrophoresis, chromatography, and the like.

[0050] In a specific embodiment of the present invention, a CottonhILIC SPE separation column is used to enrich and purify N-glycans, wherein water is used to activate the separation column, a solution of water:acetonitrile = 15:85 (volume ratio) is used to equilibrate the separation column, and pure water is used to elute the sugar chains.

[0051] In the present invention, sugar chain analysis and data processing can be performed by using analytical methods known in the art to measure and quantify sugar groups. The methods include, but are not limited to, mass spectrometry, such as matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) (such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), matrix-assisted laser desorption ionization-quadrupole ion trap-time of flight mass spectrometry (MALDI-QIT-TOF MS)), fast atom bombardment mass spectrometry (FAB-MS), electrospray mass spectrometry (ESI-MS), multistage mass spectrometry, high performance liquid chromatography HPLC, liquid chromatography-mass spectrometry (LC-MS), sugar chip technology, nuclear magnetic resonance NMR, or any combination of the above methods. Preferably, a high-resolution technique is used for analysis, such as MALDI MS.

[0052] In the present invention, the sugar chain analysis data is further calculated and processed to obtain the required sugar group related information. For example, the ratio of the peak area of ​​each sugar chain in the sample to the sum of all peak areas can be obtained, thereby obtaining the relative quantitative value of each sugar chain, which can avoid the deviation caused by the operation process such as the pretreatment of parallel samples and ensure the high accuracy of the analysis. Based on the composition characteristics and biological relevance of each sugar chain detected, derived features are calculated, including the level of fucosylation (fucosylation, F), the level of bisection sugar chains (bisection, B), the level of terminal galactosylation (galactosylation, G), the level of sialylation (sialylation, S), etc. These data can be directly used for relative content comparison or qualitative analysis to monitor the abundance changes of target sugar chains.

[0053] Sugar chain analysis data can be further calculated and processed using various related sugar chain analysis software, databases, algorithms, etc. The available sugar chain analysis software includes but is not limited to: MassyTools, Progenesis MALDI, LassyTools, GlycoWorkbench, GlycanMass, GlycoMod, GlycoFragment, GlycoSearchMS, etc. Available sugar chain analysis databases include but are not limited to: CCSD, GlycomeDB, CarbBank, EUROCarbDB, etc.

[0054] In the present invention, the sugar chain detection method is preferably a high-throughput detection method, for example, 96, 192, 288, 384 or more samples can be processed and detected simultaneously, which greatly reduces the time for sample preparation.

[0055] In the following examples, blood full sugar group detection was performed on patients with type 2 hereditary angioedema, patients with mast cell-mediated edema (disease control) and healthy controls. The cohort characteristics of the samples used are shown in Table 2. It should be noted that HAE is a rare disease. Among HAE patients in China, HAE-1 accounts for 98.73%, while HAE-2 accounts for only 1.27% (Zhi Yuxiang, An Lixin, Lai He, et al. Expert consensus on the diagnosis and treatment of hereditary angioedema [J]. Chinese Journal of Clinical Immunology and Allergy, 2019, 13 (1): 4). Therefore, the number of clinical HAE-2 cases is very limited. The cases in Table 2 are cases collected by the applicant in many years of research. For extremely rare diseases, the above sample population used in the present invention is representative, and the subsequent ROC curve analysis determined that the markers of the present invention have good diagnostic ability for type 2 hereditary angioedema.

[0056] Table 2 Baseline demographic information

[0057] Note: HAE-2: hereditary angioedema type 2, MC-AE: mast cell-mediated edema, HC: healthy control.

[0058] The above samples were used as research objects to screen plasma N-glycan markers that can be used for the diagnosis of HAE-2 and determine their clinical application value. The specific steps are as follows: 1. Glycosidase releases N-glycans N-glycans were released from whole serum / plasma glycoproteins using glycosidase PNGase F. The specific steps were as follows: 5 μL serum / plasma was taken from each sample, 10 μL 2% SDS was added, and the mixture was incubated at 60°C for 10 minutes; then 10 μL enzymatic solution (containing 2% NP-40, 2.5 × PBS and 1 U PNGase F) was added and the mixture was incubated at 37°C for 12-16 hours.

[0059] 2. N-Glycan Derivatization The free N-glycans were derivatized using known derivatization techniques, and the sialic acids of α2,3 and α2,6 linkages could be distinguished after derivatization. The specific steps are as follows: 1 μL of the above enzymatically hydrolyzed serum / plasma, add 20 μL of derivatization reagent (250 mM EDC and 250 mMhOBt, the solvent is anhydrous ethanol), and incubate at 37°C for 60 minutes.

[0060] 3. N-glycan HILIC-SPE enrichment and purification The derivatized sugar chains obtained above were enriched and purified by HILIC-SPE. HILIC uses cotton thread as the stationary phase, and the cotton thread is filled in a 20μL pipette tip to make a purification column. First, the column is activated 3 times with 15μL ultrapure water (MQ); then, the column is equilibrated with 15μL of 85% acetonitrile (ACN) 3 times; then, the derivatized sugar chain mixture is added to the column and loaded 30 times to ensure that the derivatized N-sugar chains are adsorbed on the column as completely as possible; then, the column is eluted 3 times with 15μL of 85% acetonitrile + 1% trifluoroacetic acid (TFA), and then 15μL of 85% acetonitrile 3 times; finally, the sugar chains are eluted in 10μL MQ.

[0061] 4. Mass spectrometry analysis of N-glycans Before detection, the mass spectrometer was calibrated with a peptide mixture standard of known molecular mass (Bruker Peptide Calibration Standard II). The matrix super-DHB was dissolved in a 50% acetonitrile (water) solution containing 1 mM NaOH at a concentration of 5 mg / mL. Take 1 μL of the above purified N-glycans and spot them on the mass spectrometry target plate, then add 1 μL of the matrix solution on the sample and dry it at room temperature. MALDI-TOF MS was used for analysis. The mass spectrometer was equipped with a Smartbeam 3D laser source and collected signal ions in the positive ion reflection mode (RP). FlexControl software was used for control. The m / z range was set to 1000 to 5000 during sample detection. The spectrum acquisition setting was: for each sample point on the mass spectrometry target plate, the laser completely randomly collected signals within the sample point range, accumulated 10K laser shots, collected a mass spectrum, and the laser frequency was 5000 Hz.

[0062] 5. Data preprocessing and statistical analysis The collected mass spectra were preprocessed using FlexAnalysis and MassyTools software and exported to Microsoft Excel for further analysis. The sugar chain analysis function of GlycoWorkbench was used to assist manual analysis to analyze the mass spectrometry data. The identification of sugar chain structure was mainly based on mass-to-charge ratio, secondary mass spectrometry fragment attribution and published literature. The quantification of a single sugar chain was obtained by the peak area of ​​a single sugar chain / the sum of the peak areas of all sugar chains detected. All ions were in the form of sodium addition, i.e. [M+Na] +.H = hexose (mannose or galactose); N = N-acetylglucosamine (N-acetylglucosamine: GlcNAc); F = deoxyribose (fucose); L = α2, 3-linked sialic acid; E = α2, 6-linked sialic acid. In addition to the directly detected glycan structures, derived glycan traits are calculated by Rstudio based on the directly detected N-glycans according to their structural characteristics and biological relevance. Derived glycan traits include: the number of antennae of complex-type glycans (CA), the level of fucosylation (F), the level of bisection (B), the level of terminal galactosylation (G), the level of sialylation (S), etc. Differences in N-glycosylation between HAE-2 patients and healthy controls and mast cell-mediated edema controls and the relationship between N-glycosylation features and clinical parameters were evaluated by statistical tests, regression analysis, and receiver operating characteristic curves. The quality of mass spectrometry data for the study cohort was assessed by the standards randomly distributed on the target plate during sample testing and by calculating the mean, coefficient of variation, and standard deviation of each glycan for multiple standards.

[0063] 6. Results and Discussion The average CV value of the top 30 sugar chains obtained from the quality control standard was 9.749%, indicating that the data obtained in the present invention is reliable.

[0064] Example 1 N-Glycan Markers for HAE-2 Diagnosis A total of 109 glycan structures were detected in the HAE-2 study cohort (Table 2), of which 63 glycans passed quality inspection for subsequent analysis, and the average CV value of the top 10 glycans was 5.17%. The differences in the characteristics of 63 directly detected N-glycans and 7 derived glycans in patients with hereditary angioedema type 2 (HAE-2), patients with mast cell-mediated edema (MC-AE), and healthy controls (HC) were first analyzed. The results of 3D PCA and 3D PLS-DA analysis of the total plasma N-glycan group in patients with hereditary angioedema type 2, patients with mast cell-mediated edema, and healthy controls are shown in Figure 2. Figure 1 The results of unsupervised principal component analysis showed that HAE-2 was significantly distinguished from MC-AE and HC, but MC-AE and HC were not clearly distinguished ( Figure 1 In order to further reflect the differences among the three groups, PLS-DA analysis was performed on the test data. Figure 1As shown in B, HAE-2 can be well distinguished from MC-AE and HC, while the distinction between MC-AE and HC is still not obvious, indicating that HAE-2 is significantly different from disease controls and healthy controls.

[0065] After analysis, the contents of a total of 10 N-glycan markers, including H9N2, H5N4E1, H4N5F1E1, H5N4F1L2, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, H3N4F1, and H4N4F1, were significantly different among the HAE-2, MC-AE, and HC groups. Figure 2 The box plots in the figure visualize the 10 N-glycans with significant differences among the HAE-2, MC-AE, and HC groups. The results show that there was no significant difference in plasma N-glycans between MC-AE and HC; the levels of N-glycans with fucosylation (F) were increased in HAE-2 compared with HC and MC-AE.

[0066] To identify potential N-glycan biomarkers for the diagnosis of HAE-2, the diagnostic efficacy of these markers was characterized using ROC curves ( Figure 3 Among the above 10 N-glycan markers, the AUC of H5N4E1 was 0.917, indicating a good potential for diagnosing HAE-2, and the AUCs of 7 N-glycans ranged from 0.802 to 0.899, showing good performance in diagnosing HAE-2.

[0067] Considering the influence of covariates of gender and age, the ROC after covariate adjustment was performed ( Figure 4 ). After controlling for the effects of gender and age, the AUCs of 7 out of 10 N-glycans increased, indicating that the two covariates of age and gender underestimated the diagnostic accuracy of these 7 N-glycans themselves. Among them, the AUC of H5N4E1 increased from 0.917 to 0.944, and the AUC of H9N2 increased from 0.899 to 0.911 (Table 3). On the other hand, after controlling for the effects of gender and age, the AUCs of 3 out of 10 N-glycans (H4N5F1E1, h5N4F1L2, and h3N4F1) decreased.

[0068] Table 3

[0069] However, the p values ​​for age and sex did not reach statistical significance (p>0.05), and had no significant effect on the HAE-2 diagnostic test results of the 10 N-glycans. Therefore, although these covariates had limited effects in this model, the diagnostic performance of most N-glycans was improved after controlling for the covariates.

[0070] Furthermore, the ROC of the panels composed of fucosylated (F), alpha2,6-linked sialic acid (E), and alpha2,3-linked sialic acid (L) were analyzed separately. Among them, F: sugar chain containing fucosylation; E: sugar chain containing α-2,6-linked sialic acid; L: sugar chain containing α-2,3-linked sialic acid. The F panel is H4N5F1E1, H5N4F1L2, H3N4F1, and H4N4F1; the E panel is H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, and H4N5F1E1; the L panel is H6N5E2L1, H6N5E1L1, H6N5E1L2, and H5N4F1L2.

[0071] The results show that ( Figure 5 ), the classification performance of the three panels F, E, and L in diagnosing HAE-2 was very good, and the areas under the ROC curve reached 0.994, 0.950, and 0.937, respectively. Among them, the F panel showed excellent diagnostic ability.

[0072] Similarly, covariates were adjusted for the F, E, and L panels. Figure 5 As shown, it is worth noting that after controlling for the effects of gender and age, only the AUC of the F panel among the three panels increased and increased to 1, showing a very amazing diagnostic performance in distinguishing HAE-2 from non-HAE-2 (including MC-AE and HC); in addition, the AUCs of the two panels at the E and L tails decreased slightly after controlling for covariates, but the diagnostic performance was still good.

[0073] The analysis of derived sugar chain characteristics revealed that among the derived sugar chain characteristics, the contents of seven N-sugar chain markers, including MM, CA3, CF0, CS, CG, CA2, and CF, were significantly different among the HAE-2, MC-AE, and HC groups. Figure 6 The box plots in the figure visualize the seven derived glycans with significant differences among the HAE-2, MC-AE, and HC groups. The results showed that there was no significant difference in plasma N-glycans between MC-AE and HC; compared with HC and MC-AE, the contents of the seven N-glycan markers, MM, CA3, CF0, CS, CG, CA2, and CF, were significantly changed in HAE-2.

[0074] In order to identify potential N-glycan biomarkers for the diagnosis of HAE-2, the ROC curve was used to characterize the diagnostic efficacy of the above derived glycan markers (Table 4). Among the above 7 N-glycan markers, the AUC of MM was 0.909, indicating that it has a good potential for diagnosing HAE-2, and the AUC ranges of the remaining 6 N-glycan markers were also higher than 0.8, with good HAE-2 diagnostic performance.

[0075] Considering the influence of covariates of sex and age, the ROC after covariate adjustment was performed ( Figure 7 ). After controlling for the effects of gender and age, the AUC of some N-glycans increased (Table 4), indicating that the two covariates of age and gender underestimated the diagnostic accuracy of these N-glycans themselves. However, the p-values ​​of age and gender did not reach statistical significance (p>0.05), and had no significant effect on the HAE-2 diagnostic test results of the seven N-glycans. Therefore, although the effects of these covariates in this model are limited, the diagnostic performance of most N-glycans is improved after controlling for the covariates.

[0076] Table 4

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A marker for the diagnosis of hereditary angioedema type 2, characterized in that: The markers include one or more combinations of the following sugar chain markers: H9N2, H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, H3N4F1, H4N4F1, MM, CA2, CA3, CF0, CG, CS; Alternatively, the markers include one or more selected from H4N5F1E1, H5N4F1L2, CF and one or more selected from H9N2, H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, H3N4F1, H4N4F1, MM, CA2, CA3, CF0, CG, CS.

2. The marker for diagnosing hereditary angioedema type 2 according to claim 1, characterized in that: In patients with hereditary angioedema type 2, the expression levels of H9N2, H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, MM, CA3, CF0, CS, and CG were significantly decreased compared with those in healthy subjects and patients with mast cell-mediated edema; the expression levels of H4N5F1E1, H5N4F1L2, H3N4F1, H4N4F1, CA2, and CF were significantly increased compared with those in healthy subjects and patients with mast cell-mediated edema.

3. The marker for diagnosing hereditary angioedema type 2 according to claim 1, characterized in that: The markers include H4N5F1E1, H5N4F1L2, H3N4F1 and H4N4F1; Alternatively, includes H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, and H4N5F1E1; Alternatively, includes H6N5E2L1, H6N5E1L1, H6N5E1L2 and H5N4F1L2.

4. Use of the marker for diagnosing hereditary angioedema type 2 or its detection reagent according to any one of claims 1 to 3 in the preparation of a product for diagnosing hereditary angioedema type 2.

5. The use according to claim 4, characterized in that: The product is a medicine or a kit.

6. A product for the diagnosis of hereditary angioedema type 2, characterized in that: The product comprises a marker for diagnosing hereditary angioedema type 2 or a detection reagent thereof as described in any one of claims 1 to 3.

7. The product according to claim 6, characterized in that The product is a medicine or a kit.

8. A device for diagnosing hereditary angioedema type 2, characterized in that: The device comprises: A detection module, used for detecting the content of the marker for diagnosing hereditary angioedema type 2 according to any one of claims 1 to 3 in a sample to be tested; An input module is used to obtain the detection result of the detection module; A judgment module, used to judge whether it is hereditary angioedema type 2 according to the test result obtained by the input module; The output module is used to output the judgment result.

9. The device for diagnosing hereditary angioedema type 2 according to claim 8, characterized in that: The judgment standard of the judgment module is: in patients with hereditary angioedema type 2, the expression levels of H9N2, H5N4E1, H6N5E2, H6N5E2L1, H6N5E1L1, H6N5E1L2, MM, CA3, CF0, CS, and CG are significantly decreased compared with those in healthy humans and patients with mast cell-mediated edema; the expression levels of H4N5F1E1, H5N4F1L2, H3N4F1, H4N4F1, CA2, and CF are significantly increased compared with those in healthy humans and patients with mast cell-mediated edema.

10. The device for diagnosing hereditary angioedema type 2 according to claim 8 or 9, characterized in that: The sample to be tested is plasma.

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