Plasma N-carbohydrate chain marker for severity assessment of type 2 hereditary angioedema, kit and application of plasma N-carbohydrate chain marker

Abstract

The invention relates to the technical field of medical detection, in particular to a plasma N-carbohydrate chain marker for severity assessment of type 2 hereditary angioedema, a kit and application of the plasma N-carbohydrate chain marker and the kit. The N-sugar chain marker provided by the invention is obviously related to the severity of the type 2 hereditary angioedema symptom, can be used as a marker for evaluating the severity or treatment effect of the type 2 hereditary angioedema symptom, and has relatively high specificity, sensitivity and accuracy. The N-sugar chain marker has the advantages of convenience in detection, short required time and the like when being used for evaluating the severity or treatment effect of the type 2 hereditary angioedema symptom, and has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to a plasma N-glycan marker, a kit and an application thereof for evaluating the severity of 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, resulting in delayed diagnosis of HAE.

[0003] The clinical heterogeneity of HAE is specifically reflected in the age of onset, frequency of attacks, triggering factors of acute attacks, prodromal signs, severity of edema and predilection sites. The symptoms of each attack of the same patient are also different. In mild cases, only mild acrodendrosis may occur, which does not affect normal daily life. In severe cases, if the gastrointestinal tract is involved, severe abdominal pain, nausea, vomiting, diarrhea, and abdominal effusion may occur. It is often misdiagnosed as acute abdomen, leading to unnecessary abdominal surgery. The respiratory mucosal edema caused by HAE can quickly cause dyspnea or suffocation, causing some patients to undergo multiple tracheotomies due to repeated attacks of laryngeal edema. Some patients suffocate to death due to untimely rescue. More than 50% of patients will have laryngeal edema and are at risk of death. Patients are often in a state of severe anxiety and fear, resulting in insufficient or excessive prevention, which greatly affects the quality of life and life expectancy of patients and increases the economic burden.

[0004] Chinese patent application CN113311056A discloses markers for diagnosing different degrees of severity of hereditary angioedema, but it targets HAE-1 type rather than HAE-2 type. Due to the lack of effective biomarkers for assessing the severity of HAE-2, confirmed patients often suffer from insufficient prevention leading to edema attacks or excessive prevention. Therefore, there is an urgent need for a method that can assess the severity of the disease and individual differences in HAE-2 patients, and provide a basis for doctors to formulate personalized treatment plans. For patients with severe conditions, more active treatment measures may be required to control symptoms. In addition, during the treatment process, changes in biomarkers that assess severity can also be used as indicators to evaluate the effectiveness of treatment, guiding physicians to adjust treatment plans based on changes in biomarkers to ensure maximum treatment effectiveness.

[0005] Protein glycosylation is one of the most common and complex post-translational modifications. The glycans on proteins mainly function by participating in various 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 anchor linkage, among which more than half of the glycoproteins are N-glycosylated. In recent years, the development of glycomics technology has provided a more comprehensive understanding of the biological significance of protein glycosylation in physiological and disease states. Glycomics has relatively prominent diagnostic and predictive capabilities in many aspects such as disease diagnosis, prediction of progression, and prognosis judgment. Studies have found that changes in the N-glycan profile in plasma are related to the occurrence and development of various diseases, such as lung cancer, colorectal cancer and other cancers, Alzheimer's disease, type 2 diabetes, cardiovascular diseases, etc. Summary of the Invention

[0006] The present invention provides a plasma N-glycan biomarker, a kit, and their applications for the assessment of the severity of type 2 hereditary angioedema.

[0007] The present invention provides potential biomarkers with assessment significance for the symptom severity of patients with type 2 hereditary angioedema. By scoring patients with type 2 hereditary angioedema with different symptom severities according to clinical symptoms, and detecting and analyzing the plasma whole glycome profiles of the patients, N-glycan characteristics related to clinical symptom scores and severity grading are screened, and potential biomarkers for assessing the symptom severity of patients with type 2 hereditary angioedema are determined by methods such as ROC curve analysis.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In the first aspect, the present invention provides a biomarker for the assessment of the severity of type 2 hereditary angioedema, and the biomarker comprises a combination of one or two of the following glycan biomarkers: H6N5F1E2L1, H5N4F1E1L1.

[0010] The present invention discovers that H6N5F1E2L1 and H5N4F1E1L1 show obvious correlations with the clinical symptom scores and severity grading scores of type 2 hereditary angioedema, can relatively accurately assess the severity of type 2 hereditary angioedema, and have high specificity and sensitivity.

[0011] Since the AUC of either H6N5F1E2L1 or H5N4F1E1L1 for evaluating the severity of type 2 hereditary angioedema is above 0.95, these two markers can be used alone to evaluate the severity of type 2 hereditary angioedema, and can also be used in combination. When used in combination, the AUC for evaluating the severity of type 2 hereditary angioedema must also be above 0.95, and even higher than the AUC value of each individual marker in the combination. Therefore, the combination of two of the above-mentioned glycan markers can also be used to evaluate the severity of type 2 hereditary angioedema, with high accuracy.

[0012] In some embodiments of the present invention, the marker includes one of the following glycan markers: H6N5F1E2L1, H5N4F1E1L1.

[0013] In some embodiments of the present invention, the marker includes H6N5F1E2L1 and H5N4F1E1L1.

[0014] Among the above-mentioned markers, the contents of H6N5F1E2L1 and H5N4F1E1L1 are positively correlated with the severity of type 2 hereditary angioedema.

[0015] In the present invention, the glycan marker is derived from whole plasma glycoprotein.

[0016] In the present invention, the naming rules of the glycan marker refer to the literature: Zhang Z, Westhrin M, Bondt A, Wuhrer M, Standal T, Holst S. Serum protein N-glycosylation changes in multiple myeloma. Biochim Biophys Acta Gen Subj. 2019 May;1863(5):960-970. The naming and molecular weights of these glycan structures are shown in Table 1.

[0017] Table 1

[0018] In a second aspect, the present invention provides the use of the above-mentioned marker for evaluating the severity of type 2 hereditary angioedema or its detection reagent in the preparation of a product for evaluating the severity of type 2 hereditary angioedema or the therapeutic effect of type 2 hereditary angioedema.

[0019] Preferably, the product is a drug or a kit.

[0020] In the present invention, the detection reagent for the biomarker can be any reagent for detecting the biomarker described in the present invention, for example: characteristic glycan probe, mass spectrometry detection reagent, lectin chip, etc.

[0021] In some embodiments of the present invention, the detection reagent includes a mass spectrometry detection reagent for detecting the glycan biomarker.

[0022] In some embodiments of the present invention, the detection reagent includes a characteristic glycan probe that specifically recognizes the glycan biomarker.

[0023] Optionally, the detection reagent may further include a reagent for separating plasma glycoproteins, glycosidase (such as PNGase F), N-glycan purification reagent, etc.

[0024] In a third aspect, the present invention provides the use of the biomarker for evaluating the severity of type 2 hereditary angioedema described above as a drug target for preventing or treating type 2 hereditary angioedema.

[0025] In a fourth aspect, the present invention provides a product for evaluating the severity or treatment effect of type 2 hereditary angioedema, and the product includes the biomarker for evaluating the severity of type 2 hereditary angioedema described above or its detection reagent.

[0026] Preferably, the product is a drug or a kit.

[0027] In a fifth aspect, the present invention provides a device for evaluating the severity or treatment effect of type 2 hereditary angioedema, and the device includes: A detection module for detecting the content of the biomarker for evaluating the severity of type 2 hereditary angioedema described above in a sample to be tested; An input module for obtaining the detection result of the detection module; A judgment module for evaluating the severity or treatment effect of type 2 hereditary angioedema according to the detection result obtained by the input module; An output module for outputting the judgment result.

[0028] Preferably, the judgment criterion of the judgment module is: the higher the contents of H6N5F1E2L1 and H5N4F1E1L1, the more severe type 2 hereditary angioedema is.

[0029] Preferably, the sample to be tested is plasma.

[0030] Sixthly, the present invention provides a method for evaluating the severity or treatment effect of type 2 hereditary angioedema, the method comprising: detecting the content of the above-mentioned markers for evaluating the severity of type 2 hereditary angioedema in the plasma of a subject, and evaluating the severity or treatment effect of type 2 hereditary angioedema according to the content of the markers.

[0031] Preferably, the higher the contents of H6N5F1E2L1 and H5N4F1E1L1, the more severe type 2 hereditary angioedema is.

[0032] The beneficial effects of the present invention at least include: the N-glycan markers provided by the present invention are significantly correlated with the severity of type 2 hereditary angioedema symptoms, and can be used as markers for evaluating the severity or treatment effect of type 2 hereditary angioedema symptoms, having high specificity, sensitivity and accuracy. Using the N-glycan markers of the present invention to evaluate the severity or treatment effect of type 2 hereditary angioedema symptoms also has advantages such as convenient detection and short required time, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 It is the correlation between the content change of plasma N-glycans and the severity score of type 2 hereditary angioedema in Example 1 of the present invention.

[0035] Figure 2 It is the correlation heat map between the glycan markers and the severity of type 2 hereditary angioedema in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] In order to make the objectives, technical solutions and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention with reference to the drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments in the present invention belong to the scope of protection of the present invention.

[0037] The "glycome" as mentioned in the present invention refers to all the glycans expressed in a sample (such as body fluid, cell, tissue) or all the glycans on a certain type of specific glycoprotein.

[0038] 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.

[0039] The sugar chains described in the present invention may be free sugar chains or sugar chains released from sugar complexes.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] In a specific embodiment of the present invention, a Cotton HILIC 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.

[0044] 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.

[0045] In the present invention, the glycan analysis data is further calculated and processed to obtain the required glycome-related information. For example, the ratio of the peak area of each glycan in the sample to the sum of all peak areas can be obtained, so as to obtain the relative quantitative value of each glycan, which can avoid the deviation caused by the pretreatment of parallel samples and ensure the high accuracy of the analysis; according to the composition characteristics and biological relevance of each detected glycan, derived features are calculated, including the level of fucosylation (F), the level of bisecting glycan (B), the level of terminal galactosylation (G), the level of sialylation (S), etc. These data can be directly used for relative content comparison or qualitative analysis to monitor the changes in the abundance of target glycans or glycan-derived features.

[0046] For the further calculation and processing of the glycan analysis data, various relevant glycan analysis software, databases, algorithms, etc. can be used to analyze the obtained data. The available glycan analysis software includes but is not limited to: MassyTools, Progenesis MALDI, LassyTools, GlycoWorkbench, GlycanMass, GlycoMod, GlycoFragment, GlycoSearchMS, etc. The available glycan analysis databases include but are not limited to: CCSD, GlycomeDB, CarbBank, EUROCarbDB, etc.

[0047] In the present invention, the detection method of glycans 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 sample preparation time.

[0048] In the following examples, whole glycome detection of clinical samples was performed. The cohort characteristics of the samples used are shown in Table 2. It should be noted that HAE is a rare disease. Among Chinese HAE patients, type HAE-1 accounts for 98.73%, while type HAE-2 only accounts for 1.27% (Yu-Xiang Zhi, Li-Xin An, He Lai, 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 cases of type HAE-2 is very limited. The cases in Table 2 are the 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 subsequent ROC curve analysis determined that the markers of the present invention have good predictive ability for the severity of type 2 hereditary angioedema symptoms.

[0049] Table 2 Demographic baseline information

[0050] Note: HAE-2: Hereditary angioedema type 2.

[0051] In order to distinguish HAE-2 patients with different severities, the present invention first scores the clinical symptoms of HAE-2 patients (Table 3). Two HAE clinical symptom scores are used, evaluated in the form of numbers and grades respectively. In Scoring Scale 1, the average severity score of 12 patients is 4.42, and in Scoring Scale 2, more than half of the HAE-2 patients have severe severity. In order to reduce the severity result bias caused by the different focuses of the two scales, a correlation analysis of the two clinical symptom scores was first performed, and the results showed that the two clinical symptom scores were significantly correlated (r = 0.847, p < 0.01, Figure 1 of A).

[0052] Table 3 Patient clinical symptom and severity information

[0053] Taking the above samples as the research objects, plasma N-glycan markers that can be used to evaluate the severity of hereditary angioedema type 2 are screened and their clinical application values are judged. The specific steps are as follows: 1. Release of N-glycans by glycosidase Release N-glycans from whole serum / plasma glycoproteins by glycosidase PNGase F. The specific steps are as follows: Take 5 μL of serum / plasma from each sample, add 10 μL of 2% SDS, incubate at 60 °C for 10 minutes; then add 10 μL of enzymatic hydrolysis solution (containing 2% NP-40, 2.5 × PBS and 1 U PNGase F), and incubate at 37 °C for 12 - 16 h.

[0054] 2. Derivatization of N-glycans Derivatize the N-glycans obtained above by known derivatization techniques. After derivatization, sialic acids with α2,3 and α2,6 linkages can be distinguished. The specific steps are as follows: Add 1 µL of the above enzymatically hydrolyzed serum / plasma to 20 µL of derivatization reagent (250 mM EDC and 250 mM HOBt, solvent is absolute ethanol), and incubate at 37 °C for 60 minutes.

[0055] 3. HILIC-SPE enrichment and purification of N-glycans The derivatized glycan obtained above was enriched and purified by HILIC-SPE. HILIC uses cotton thread as the stationary phase. The cotton thread was self-packed into a 20 µL pipette tip to make a purification column. First, the column was activated 3 times with 15 µL of ultrapure water (MQ); then, the column was equilibrated 3 times with 15 μL of 85% acetonitrile (ACN); then the above derivatized glycan mixture was added to the column and loaded 30 times to ensure that the derivatized N-glycans were adsorbed on the column as completely as possible; then the column was washed 3 times with 15 μL of 85% acetonitrile + 1% trifluoroacetic acid (TFA), and then washed 3 times with 15 μL of 85% acetonitrile; finally, the glycan was eluted into 10 μL of MQ.

[0056] 4. Mass spectrometry analysis of N-glycans Before detection, the mass spectrometry instrument 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-glycan and spot it on the mass spectrometry target plate, then add 1 µL of the matrix solution dropwise on the sample and dry it at room temperature. MALDI-TOF MS was used for analysis. The mass spectrometry was equipped with a Smartbeam 3D laser source, and signal ions were collected in the positive ion reflection mode (reflection positive, RP). The FlexControl software was used for control. The m / z range was set to 1000 to 5000 during sample detection. The spectrum acquisition was set as follows: for each sample spot on the mass spectrometry target plate, the laser randomly collected signals within the range of this sample spot, accumulated 10K laser shots, and collected a mass spectrometry spectrum. The laser frequency was 5000 Hz.

[0057] 5. Data preprocessing and statistical analysis The collected mass spectrometry spectra were preprocessed using FlexAnalysis and MassyTools software and exported to Microsoft Excel for further analysis. The glycan parsing function of GlycoWorkbench was used to assist manual parsing to analyze the mass spectrometry data. The identification of glycan structures was mainly based on the mass-to-charge ratio, secondary mass spectrometry fragment attribution, and published literature. The quantification of individual glycans was obtained by the peak area of individual glycans / the sum of the peak areas of all detected glycans. All ion forms were sodium adducts, i.e., [M+Na]. +.H = hexose (mannose or galactose); N = N-acetylhexosamine (N-acetylglucosamine: GlcNAc); F = deoxyribose (fucose); L = α2,3-linked sialic acid; E = α2,6-linked sialic acid. The differences in N-glycosylation among HAE-2 patients with different severities and the relationship between N-glycosylation characteristics and clinical parameters were evaluated by statistical tests, regression analysis, and receiver operating characteristic curves. The quality of the mass spectrometry data of the study cohort was evaluated by the standards randomly distributed on the target plate during the sample detection process and the calculated average value, coefficient of variation, and standard deviation of each sugar chain of multiple standards.

[0058] 6. Results and Discussion The average CV value of the top 30 sugar chains obtained from the quality control standards was 9.749%, indicating that the data obtained in the present invention is reliable.

[0059] Example 1 N-Glycan Markers for the Assessment of the Severity of HAE-2 A total of 109 sugar chain structures were detected in the HAE-2 study cohort (Table 2), among which 63 sugar chains passed the quality test for subsequent analysis, and the average CV value of the top 10 sugar chains was 5.17%. The differences in the 63 directly detected N-glycans among HAE-2, MC-AE, and HC were analyzed for the first time. The sugar chain characteristics related to two symptom scores were analyzed separately. In symptom score scale 1, a total of 5 sugar chains (H6N5F1E2L1, H6N5F1E1L2, H5N4F1E1L1, H5N4E2, H7N6E2L2) were significantly correlated with it ( Figure 1 B-F), among which, the 4 sugar chains H6N5F1E2L1, H6N5F1E1L2, H5N4F1E1L1, and H5N4E2 were positively correlated with symptom score scale 1, and H7N6E2L2 was negatively correlated with score scale 1. In symptom score scale 2, 3 sugar chains (H5N4F1E1L1, H6N5F1E2L1, H4N4) were significantly positively correlated with it ( Figure 1 G-I); among them, H5N4F1E1L1 and H6N5F1E2L1 were significantly positively correlated with both score scale 1 and score scale 2 (Table 4). The correlation heat map ( Figure 2 ) further verified the Figure 1 correlation analysis. In symptom score scale 1, 5 sugar chains (H6N5F1E2L1, H6N5F1E1L2, H5N4F1E1L1, H5N4E2, H7N6E2L2) were significantly correlated with it, and in symptom score scale 2, 3 sugar chains (H5N4F1E1L1, H6N5F1E2L1, H4N4) were significantly correlated with it.

[0060] After that, by evaluating the glycans related to severity, the results of independent sample t-tests and receiver operating characteristic (ROC) curve results showed that H5N4F1E1L1 and H6N5F1E2L1 were significantly correlated with patients with a score of ≥7 in symptom rating scale 1 and were significantly correlated with patients with severe symptoms in symptom rating scale 2 (Table 5).

[0061] Table 4 Table 5

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A marker for evaluating the severity of hereditary angioedema type 2, characterized in that, The marker includes one or a combination of two of the following glycan markers: H6N5F1E2L1, H5N4F1E1L1.

2. The biomarker for evaluating the severity of type 2 hereditary angioedema according to claim 1, wherein The contents of H6N5F1E2L1 and H5N4F1E1L1 are positively correlated with the severity of hereditary angioedema type 2.

3. The biomarker for evaluating the severity of type 2 hereditary angioedema according to claim 1 or 2, characterized in that, The glycan marker is derived from whole plasma glycoprotein.

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

5. The application according to claim 4, wherein The product is a drug or a kit.

6. A product for evaluating the severity or treatment effect of hereditary angioedema type 2, characterized in that, The product includes the marker or its detection reagent for evaluating the severity of hereditary angioedema type 2 according to any one of claims 1 to 3.

7. The product according to claim 6, wherein The product is a drug or a kit.

8. A device for evaluating the severity or treatment effect of type 2 hereditary angioedema, characterized in that, The device includes: A detection module for detecting the content of the marker for evaluating the severity of hereditary angioedema type 2 according to any one of claims 1 to 3 in a sample to be tested; An input module for obtaining the detection result of the detection module; A judgment module for evaluating the severity or therapeutic effect of hereditary angioedema type 2 according to the detection result obtained by the input module; An output module for outputting the judgment result.

9. The device for evaluating the severity of type 2 hereditary angioedema according to claim 8, wherein, The judgment criterion of the judgment module is: the higher the contents of H6N5F1E2L1 and H5N4F1E1L1, the more severe hereditary angioedema type 2 is.

10. The device for evaluating the severity or treatment effect of type 2 hereditary angioedema according to claim 8 or 9, characterized in that, The sample to be tested is plasma.

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