Markers for assessing severity of alopecia

Abstract

The invention provides application of a reagent and / or an instrument for detecting an N-sugar chain on IgG protein in a sample in preparation of a product for evaluating the severity of alopecia of a subject.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to markers for assessing the severity of hair loss. Background Art

[0002] Androgenic alopecia is the most common type of hair loss in men. With the improvement of living standards, hair loss and hair loss anxiety are becoming increasingly common. Androgenic alopecia is mainly caused by genetics and androgens. Affected by genetic, hormonal and other factors, the signal pathways in the hair follicle cells of patients are dysregulated, triggering an abnormal inflammatory response, resulting in a shortened hair growth period and miniaturization of hair follicles - that is, the terminal hair becomes thinner and softer, and is eventually replaced by vellus hair. Androgenic alopecia is characterized by youthfulness and gradual progression. Early identification and treatment can prevent deterioration and preserve hair as much as possible, and most patients require long-term treatment to maintain the effect. Therefore, finding biomarkers that predict the severity of androgenic alopecia will help with early prevention and intervention in risk groups.

[0003] Changes in IgG surface N-glycan content are associated with aging, the clinical classification, progression, and severity of tumors, neurodegenerative diseases, metabolic diseases, autoimmune diseases, and inflammatory diseases. Currently, there are no reports in this field correlating IgG surface N-glycan content with the severity of hair loss. Summary of the Invention

[0004] In response to the above technical problems, the present invention studies the differences in the content of N-glycans on IgG proteins in patients with different hair loss severity, and determines that H5N4F1S2 on the IgG surface can be used to predict the severity of hair loss.

[0005] In a first aspect, the present invention provides the use of a reagent and / or apparatus for detecting N-glycans on IgG proteins in a sample, in the preparation of a product for assessing the severity of hair loss in a subject. The N-glycans are H5N4F1S2, where H represents a hexose, N represents N-acetylglucosamine, F represents fucose, and S represents sialic acid. H5 represents five hexoses, N4 represents four N-acetylglucosamines, F1 represents one fucose, and S2 represents two sialic acids.

[0006] In some embodiments, the H5N4F1S2 has Figure 1 The structure shown.

[0007] In some embodiments, the subject is a patient with androgenic alopecia; in some embodiments, the subject is a patient with male androgenic alopecia; in some embodiments, the subject is a patient with male androgenic alopecia that exhibits characteristics of female androgenic alopecia (male FPHL).

[0008] In some embodiments, the sample is blood, plasma, or serum.

[0009] In some embodiments, the reagents and / or instruments include reagents and / or instruments used in a method selected from the following: mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry, sugar chip technology or nuclear magnetic resonance; preferably, the method is ultra-high performance liquid chromatography.

[0010] In some embodiments, the reagents and / or instruments further include one or more reagents and / or instruments selected from the following: reagents and / or instruments for collecting and / or processing samples; reagents and / or instruments for separating and / or purifying proteins in samples; reagents and / or instruments for separating, purifying and / or enriching N-glycans on proteins in samples.

[0011] A second aspect of the present invention provides a system for assessing the severity of hair loss in a subject, comprising: 1) a reagent for detecting the level of N-sugar chain H5N4F1S2 on IgG protein in a sample, wherein H represents hexose, N represents N-acetylglucosamine, F represents fucose, and S represents sialic acid; and 2) a device comprising a data input module, a data comparison module, and a conclusion output module.

[0012] In some embodiments, the subject is a patient with androgenic alopecia; preferably a patient with male androgenic alopecia; more preferably a patient with male androgenic alopecia showing features of female androgenic alopecia (male FPHL).

[0013] In some embodiments, the sample is blood, plasma, or serum.

[0014] In some embodiments, the reagent comprises a reagent used in a method selected from the group consisting of mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry, carbohydrate chip technology, or nuclear magnetic resonance; preferably, the method is ultra-high performance liquid chromatography.

[0015] In some embodiments, the reagents further include one or more reagents selected from the following: reagents for collecting and / or processing samples; reagents for separating and / or purifying proteins in samples; reagents for separating, purifying and / or enriching N-glycans on proteins in samples.

[0016] The advantages of the present invention over the prior art are:

[0017] This study demonstrates for the first time that H5N4F1S2 on the surface of IgG is significantly associated with the severity of hair loss in patients with male androgenetic alopecia (FPHL), a condition characterized by female pattern hair loss. Specifically, H5N4F1S2 is negatively correlated with the severity of hair loss. Evaluations using test and validation datasets demonstrate that using H5N4F1S2 on the surface of IgG to assess hair loss severity has high specificity, sensitivity, and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] By reading the following detailed description of non-limiting embodiments with reference to the accompanying drawings, other features, objects and advantages of the present invention will become more apparent.

[0019] Figure 1 An example structure of H5N4F1S2 is shown.

[0020] Figure 2 Plot showing Pearson correlation analysis between FPHL severity and measured IgG surface H5N4F1S2 levels in males in the test set.

[0021] Figure 3 ROC curves showing the severity of FPHL and the measured IgG surface H5N4F1S2 in males in the test set.

[0022] Figure 4 Figure 2. Spearman correlation analysis of the severity of FPHL in males in the training set and the measured IgG surface H5N4F1S2 levels.

[0023] Figure 5 ROC curves showing the severity of FPHL and the measured IgG surface H5N4F1S2 in males in the training set. DETAILED DESCRIPTION

[0024] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described 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.

[0025] Unless otherwise defined, technical or scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0026] Example 1 Discovery of Male FPHL Hair Loss Severity Markers

[0027] In accordance with the principle of informed consent, a total of 23 patients aged 18-60 years with male androgenic alopecia (male FPHL) showing features of female androgenic alopecia who visited the outpatient clinic of Huashan Hospital Affiliated to Fudan University between June and December 2022 were recruited. Each patient's age, course of disease, family history, and other medical history were recorded, and at least three dermatologists assessed the severity of hair loss based on the patient's clinical photographs according to the Sinclair grading system. Sinclair grades 2-3 are defined as mild to moderate hair loss, and grades 4-5 are defined as severe hair loss.

[0028] Collect venous blood samples from patients for glycomics research. The specific procedures are as follows:

[0029] (1) Obtaining serum samples from male FPHL patients: Collect 5 ml of peripheral venous blood from the subject and centrifuge at 4°C, 3000 rpm, for 8 minutes. Collect the upper layer of serum, taking care not to collect the lower layer of red blood cells.

[0030] (2) Purification of IgG: Add 200 μl of binding buffer (1×PBS) to the purification column, slowly pass through the column, and equilibrate the purification column. Mix 50 μl of binding buffer with 50 μl of serum sample, load the sample onto the equilibrated purification column, incubate at room temperature for 1 hour, centrifuge, and repeat the loading three times. Then elute the IgG with 400 μl of elution buffer (100 mM glycine pH = 2.5), collect the eluate, and neutralize it with 40 μl of pH-adjusted liquid Tris-HCl to obtain purified IgG.

[0031] (3) Release of IgG N-glycans: Take 100 μl of purified IgG, add 1 μl of PNGase F endoglycosidase (New England Biolabs, USA, P0705L), mix thoroughly, and incubate at 37°C for 12 hours.

[0032] (4) Enrichment of IgG N-glycans: Activate the porous graphitized carbon on a 96-well PVDF membrane filter plate with a mixed solution of 80% acetonitrile and 0.1% trifluoroacetic acid, and then equilibrate the porous graphitized carbon column with a 0.1% trifluoroacetic acid solution. Add the N-glycan enzymatic hydrolysis mixture to be enriched to the equilibrated porous graphitized carbon column and repeat the loading three times. Add H2O to wash away the salt and buffer. Elute the N-glycans with a mixed solution of 25% acetonitrile and 0.05% trifluoroacetic acid. Concentrate and dry the eluate in a spin dryer for 3 hours (45°C).

[0033] (5) Labeling IgG N-glycans: Label IgG N-glycans with 2-aminobenzamide. Prepare a labeling mixture by dissolving 50 mg of 2-aminobenzamide and 60 mg of sodium cyanoborohydride in 1 ml of a mixture of glacial acetic acid and dimethyl sulfoxide. Add 3 μl of the labeling mixture to each sample, shake and mix thoroughly, and incubate at 60°C for 2 hours. Terminate the labeling reaction with 50 μl of H2O. Store the 2-aminobenzamide-labeled IgG N-glycan samples in a refrigerator at 4°C.

[0034] (6) Quantitative Detection of IgG N-Glycans by Ultra-High Performance Liquid Chromatography: Analysis was performed using a Nexera UHPLC LC-30A. Mobile phase A was 100 mmol / L ammonium formate at pH 4.4, and mobile phase B was 100% acetonitrile. The fluorescence detector was set at an excitation wavelength of 330 nm and an emission wavelength of 420 nm. Separation was performed using an amino column at a column temperature of 70°C, a flow rate of 0.5 ml / min, and a linear gradient.

[0035] (7) Data processing: The chromatograms were preprocessed using LabSolutions software. The content of a single component = the area of ​​each peak / the total area of ​​all peaks.

[0036] (8) IBM SPSS Statistics 25 and GraphPad Prism 9 were used to perform statistical analysis of the data. Non-normally distributed quantitative data were represented by median and quartiles (first quartile, third quartile); normally distributed quantitative data were represented by mean ± standard deviation. The Mann-Whitney U test or independent sample t-test was used for comparison between the two groups. P < 0.05 indicated that the difference was statistically significant. Receiver operating characteristic (ROC) curves were used to compare the sensitivity and specificity of N-glycan prediction performance, and the area under the curve (AUC) was used to evaluate the accuracy. The higher the AUC value, the better the diagnostic efficacy.

[0037] Table 1. Comparison of serum immunoglobulin G (IgG) N-glycan content in male FPHL patients with different disease severity grades [%, x ± s]

[0038] IgG N-glycans Mild to moderate hair loss group (13 cases) Severe hair loss group (10 cases) t-value P-value H5N4F1S2 2.94±0.29 2.47±0.24 4.197 0.0004

[0039] Pearson correlation analysis was performed on the hair loss grade of male FPHL patients and the expression levels of 23 IgG surface N-glycans. The results showed that the IgG surface N-glycan H5N4F1S2 was significantly correlated with the hair loss grade of male FPHL patients (Table 1). As the severity of hair loss increased, the content of H5N4F1S2 decreased (correlation coefficient r = -0.675, P = 0.0004). Figure 2 As shown in Figure 2, the ROC curve shows that IgG surface H5N4F1S2 has a high accuracy in predicting the degree of hair loss in male FPHL (AUC = 0.92, P = 0.0008). Figure 3 H5N4F1S2 had a sensitivity of 90% and a specificity of 84.6%, indicating that IgG surface H5N4F1S2 can be used as a biomarker to predict the severity of hair loss in male FPHL patients. For example, a blood IgG surface H5N4F1S2 level greater than 2.68% indicates mild to moderate hair loss, while a blood IgG surface H5N4F1S2 level ≤ 2.68% indicates severe hair loss.

[0040] Example 2: Validation of H5N4F1S2 as a marker of hair loss severity in male FPHL patients

[0041] Sixteen male patients with androgenetic alopecia (FPHL) aged 18-60 years who presented at the outpatient clinic of Huashan Hospital Affiliated to Fudan University between June and December 2022 were selected as a validation set to verify the feasibility of H5N4F1S2 as a marker for hair loss severity in male FPHL patients. For hair loss severity assessment and glycomics research methods, see Example 1.

[0042] Spearman correlation analysis was performed on the hair loss grade of male FPHL patients and the expression levels of 23 IgG surface N-glycans. The results showed that the IgG surface N-glycan H5N4F1S2 was significantly correlated with the hair loss grade of male FPHL patients. As the severity of hair loss increased, the content of H5N4F1S2 decreased (correlation coefficient r = -0.687, P = 0.0055). Figure 4 shown.

[0043] According to the ROC curve, IgG surface H5N4F1S2 has a high accuracy in predicting the degree of hair loss in male FPHL (AUC = 0.93, P = 0.0078). Figure 5 Among them, H5N4F1S2 had a sensitivity of 80% and a specificity of 100%, indicating that IgG surface H5N4F1S2 can be used as a biomarker to predict the severity of hair loss in male FPHL patients.

[0044] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, from all points of view, the embodiments should be regarded as illustrative and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and range of equivalents of the claims are included in the present invention. Any reference signs in the claims should not be construed as limiting the claim to which they relate.

[0045] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Use of a reagent and / or instrument for detecting N-glycans on IgG proteins in a sample in the preparation of a product for assessing the severity of hair loss in a subject, characterized in that: The N-sugar chain is H5N4F1S2, wherein H represents hexose, N represents N-acetylglucosamine, F represents fucose, and S represents sialic acid.

2. The method according to claim 1, wherein the subject is a patient with androgenic alopecia; preferably, the subject is a patient with male androgenic alopecia; preferably, the subject is a patient with male androgenic alopecia (male FPHL) that exhibits characteristics of female androgenic alopecia.

3. The use according to claim 1 or 2, wherein the sample is blood, plasma or serum.

4. The use according to any one of claims 1 to 3, wherein the reagents and / or instruments include reagents and / or instruments used in a method selected from the group consisting of mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry, carbohydrate chip technology, or nuclear magnetic resonance; preferably, the method is ultra-high performance liquid chromatography.

5. The use according to any one of claims 1 to 4, further comprising one or more reagents and / or instruments selected from the following: reagents and / or instruments for collecting and / or processing samples; Reagents and / or instruments used to separate and / or purify proteins from samples; Reagents and / or instruments used to separate, purify, and / or enrich N-glycans on proteins in a sample.

6. A system for assessing the severity of hair loss in a subject, comprising: 1) a reagent for detecting the level of N-glycan H5N4F1S2 on IgG protein in a sample, wherein H represents hexose, N represents N-acetylglucosamine, F represents fucose, and S represents sialic acid; and 2) an apparatus comprising a data input module, a data comparison module, and a conclusion output module.

7. The system according to claim 6, wherein: The subject is a patient with androgenic alopecia; preferably a patient with male androgenic alopecia; more preferably a patient with male androgenic alopecia (male FPHL) showing characteristics of female androgenic alopecia.

8. The system according to claim 6 or 7, wherein: The sample is blood, plasma or serum.

9. The system according to any one of claims 6 to 8, wherein the reagents include reagents used in a method selected from the group consisting of mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry, carbohydrate chip technology, or nuclear magnetic resonance; preferably, the method is ultra-high performance liquid chromatography. The system according to any one of claims 6 to 9, wherein the reagents further comprise one or more reagents selected from the following: reagents for collecting and / or processing samples; reagents for separating and / or purifying proteins in samples; reagents for separating, purifying and / or enriching N-glycans on proteins in samples.

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