Biomarkers for the classification and diagnosis of atypical depression and their applications

Abstract

The present invention relates to biomarkers for detecting atypical depression, and provides an application method for detecting differential expression levels of CD23 and PTX3 proteins as plasma biomarkers for atypical depression. The method of the present invention is used for subtype classification diagnosis of atypical depression for non-diagnostic purposes, thereby improving the sensitivity and specificity of atypical depression screening.

Description

Technical Field

[0001] The present invention relates to the technical field of clinical medicine psychiatry, more specifically, to the technical field of depression, and in particular to a subtype classification-assisted diagnosis and detection technology for determining atypical depression. Background Art

[0002] Major depressive disorder is widely recognized as a heterogeneous disease. This heterogeneity stems primarily from differences in disease characteristics, diverse development and evolution, and individual variations, a characteristic also reflected in diagnostic criteria. The diagnostic criteria for depression in the Diagnostic and Statistical Manual of Mental Disorders (5th edition) (DSM-5) are based on a minimum number of symptom features within a multivariate symptom profile (at least five of the nine included symptom features, with at least one of these features being "low mood" or "diminished interest or pleasure"). This diagnostic criteria allows for at least 227 different symptom combinations to occur across different patients with depression. Considering that some diagnostic items are multivariate, such as "weight gain or loss" and "psychomotor agitation or retardation," it is estimated that there are as many as 14,528 possible symptom combinations across patients.

[0003] The heterogeneity of depression leads to great differences in symptom patterns, disease course and treatment responses among patients. Therefore, based on homogeneous patient subgroups or subtypes, it is helpful to reduce the heterogeneity among depression patients, and to optimize the treatment plan based on the patient's clinical characteristics, thereby achieving "individualized treatment" of depression, improving the diagnosis and treatment level of depression, and helping the patient's prognosis and social function recovery. Therefore, the accurate identification of specific clinical subtypes with certain characteristics among depression patients is of extremely important clinical significance.

[0004] Regarding clinical subtypes of depression, the three most common "pure" subtypes are anxious depression, melancholic depression, and atypical depression. Atypical depression affects approximately 15-50% of the depressed population. Its main clinical features, in addition to the core features of depression, include pronounced emotional reactivity, severe fatigue, anxiety, significant weight or appetite gain, increased sleepiness, leaden paralysis (a feeling of intense sleepiness), and chronic interpersonal rejection sensitivity. Atypical depression typically develops at an earlier age, often in the 20s or even earlier, and has a protracted course, high comorbidity rates, and a high risk of suicide. In particular, it is associated with a poor response to antidepressant medication and a high risk of transitioning to mania. Approximately 45.4% of patients with atypical depression subsequently develop bipolar disorder, and they are also more likely to develop long-term, chronic depression. However, clinical identification of atypical depression subtypes is difficult, and biological identification offers greater precision. Therefore, the development of objective biomarkers for the precise identification of atypical depression is of paramount clinical value. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for subtype classification diagnosis of atypical depression in response to the insufficient recognition of different subtypes of depression in the existing technology: that is, by detecting the differential expression levels of Fcε receptor II (Fc epsilon receptor II, FER2, also known as CD23) and pentraxin 3 (Pentraxin 3, PTX3) proteins, providing clinically practical and effective objective biological indicators, thus becoming a method for subtype classification diagnosis of atypical depression.

[0006] To achieve the above objectives, the present invention provides an application of a biomarker in the preparation of a product for screening or subtype classification diagnosis of depression, wherein the biomarker is one or both of CD23 and PTX3 proteins.

[0007] Preferably, the product is a product for screening atypical depression.

[0008] Preferably, the product is a product for subtype classification and diagnosis of atypical depression.

[0009] The present invention also provides a use of a reagent for detecting the content of a biomarker in preparing a kit for screening or subtype classification diagnosis of depression, wherein the main feature is that the biomarker is one or both of CD23 and PTX3 proteins.

[0010] The present invention also provides a kit for use in the screening or subtype classification diagnosis of depression, the main feature of which is that the kit is used to detect the content of biomarkers, and the biomarkers are one or both of CD23 and PTX3 proteins.

[0011] The present invention provides an application method for detecting differential expression levels of one or both of the CD23 and PTX3 proteins as plasma biomarkers for atypical depression. The method of the present invention can be used to screen for depression, particularly atypical depression. Furthermore, it can also be used for subtype classification and diagnosis of atypical depression for non-diagnostic purposes, thereby improving the sensitivity and specificity of atypical depression screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the overall process for subtype classification and diagnosis of atypical depression by detecting the differential expression levels of CD23 and PTX3 proteins.

[0013] Figure 2 Figure 3. Comparison of plasma CD23 and PTX3 protein expression levels (pg / ml) in melancholic depression, atypical depression, anxious depression, and healthy controls. The center point is the mean, and the error bars are the standard error. ** indicates that the CD23 and PTX3 protein expression levels in the other groups were statistically significantly different from those in the atypical depression group, i.e., P < 0.01 after correction.

[0014] Figure 3 Figure 2 shows receiver operating characteristic (ROC) curves for plasma CD23 protein in differentiating patients with atypical depression from healthy controls, melancholic depression, and anxious depression. (A) ROC curves for plasma CD23 protein in differentiating patients with atypical depression from healthy controls (A), melancholic depression (B), and anxious depression (C). The abscissa represents 1-specificity, the ordinate represents sensitivity, and the AUC (Area Under Curve) represents the area under the ROC curve.

[0015] Figure 4 Figure 3. ROC curves for plasma PTX3 protein in differentiating atypical depression from healthy controls, melancholic depression, and anxious depression. (A) ROC curves for plasma PTX3 protein in differentiating atypical depression from healthy controls (A), melancholic depression (B), and anxious depression (C). The abscissa represents 1-specificity, the ordinate represents sensitivity, and the AUC (Area Under Curve) represents the area under the ROC curve. DETAILED DESCRIPTION

[0016] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than for limiting the claims of the invention.

[0017] Considering that depression is a highly clinically heterogeneous disease, with significant heterogeneity in clinical symptoms, treatment strategies, treatment effects, prognosis, and even biological phenotypes, the present invention provides a method for diagnosing atypical depression, a clinical subtype with a large number of patients, particularly adolescent depression, by detecting one or both of the CD23 and PTX3 proteins. This method can be used to diagnose atypical depression and healthy controls, as well as clinical subtypes such as anxious depression and melancholic depression. Providing objective markers for the classification and diagnosis of atypical depression, an important subtype, will guide the clinical diagnosis and treatment efficacy prediction of patients with this depressive subtype, thereby providing a reference value for the auxiliary diagnosis of atypical depression. This method can also screen out patients with depression with atypical characteristics, thereby prompting and assisting treating physicians in adopting appropriate treatment plans and improving patients' treatment plans, outcomes, and prognosis.

[0018] Protein chip technology can be used to detect one or both of the patient's CD23 and PTX3 proteins. On the one hand, it can screen out patients with atypical depression subtypes from patients with different subtypes of depression, such as atypical depression, anxious depression, melancholic depression, and healthy controls, thereby serving as an objective biological indicator for auxiliary diagnosis of atypical depression. On the other hand, it can also be used in patients diagnosed with depression through routine psychiatric examinations to further confirm the diagnosis and subtype classification of patients diagnosed with depression through psychiatric examinations, thereby improving the accuracy of clinicians' diagnosis, avoiding false positives, and improving disease prognosis.

[0019] Therefore, by detecting the differential expression levels of one or both of the plasma CD23 and PTX3 proteins, a diagnostic hint for an atypical depression subtype can be obtained, which can be directly provided to clinicians for reference, enabling them to determine an accurate treatment plan in a shorter period of time, thereby shortening the diagnosis and treatment time and improving work efficiency and the accuracy of the diagnosis and treatment plan.

[0020] In the present invention, the biomarkers are CD23 and PTX3 proteins, which are suitable for the classification diagnosis of atypical depression.

[0021] In the present invention, the terms "depression", "depressive symptoms", "depressive episode" and their meanings are as follows:

[0022] Major depressive disorder is a general term for a class of diseases characterized by low mood or depressed state of mind, accompanied by varying degrees of cognitive and behavioral changes, and may be accompanied by psychotic symptoms such as hallucinations and delusions. It is characterized by high prevalence, high recurrence rate, high suicide rate and high disability. Currently, clinical diagnosis of depression mainly adopts classification diagnostic systems such as the International Classification of Diseases, 10th edition (ICD-10) and the Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5).

[0023] A major depressive episode is a condition characterized by depression, with low mood, loss of interest, and anhedonia as core features, often accompanied by other cognitive, physical, and behavioral manifestations, such as inattention, slowed response, sleep disturbances, decreased activity, and fatigue. A single depressive episode lasts at least two weeks and often recurs. While most episodes resolve, some may have residual symptoms or become chronic, potentially causing serious impairment in social functioning.

[0024] Depressive symptoms refer to the clinical manifestations of a depressive episode and can be broadly categorized into core, psychological, and somatic symptoms. Core symptoms include low mood, loss of interest, and anhedonia; psychological symptoms include anxiety, slowed thinking, cognitive symptoms, self-blame, suicidal attempts and behaviors, psychomotor retardation or agitation, psychotic symptoms, and loss of insight; and somatic symptoms include sleep disorders, eating and weight disorders, loss of energy, depressive mood that worsens during the day and lessens at night, sexual dysfunction, and other nonspecific somatic symptoms.

[0025] In the present invention, CD23 and PTX3 proteins are both human proteins and are expressed in plasma. Their basic information and functions are as follows:

[0026] CD23, encoded by the FCER2 gene, is expressed exclusively in adipose tissue. Mutations in this gene are associated with adiponectin deficiency. CD23 is a 45-kDa glycoprotein located on the surface of B lymphocytes. It serves as a differentiation antigen and surface marker for B lymphocytes, typically expressed on activated B cells in germinal centers. It has multiple functions, including mediating B lymphocyte activation and differentiation, and processing and presenting antigens. Furthermore, it serves as a low-affinity receptor for immunoglobulin E (IgE) and CR2 / CD21. It plays an important role in regulating IgE production and B cell differentiation (as a B cell-specific antigen). CD23 is primarily distributed intracellularly and can be secreted into the blood. It may circulate in plasma and participate in metabolic and hormonal regulation.

[0027] PTX3 is a long pentapeptide-related protein whose expression is induced by inflammatory cytokines in response to inflammatory stimuli in several mesenchymal and epithelial cell types, particularly endothelial cells and mononuclear phagocytes. PTX3 promotes fibrocyte differentiation and is involved in regulating inflammation and complement activation. It also plays a role in angiogenesis and tissue remodeling. This protein serves as a biomarker for various inflammatory conditions. Furthermore, it plays a role in regulating innate resistance to pathogens, inflammatory responses, possible clearance of autologous components, and female fertility.

[0028] The term "ROC curve" (receiver operator characteristic curve) used in the present invention refers to a graphical curve that shows the performance of a binary classification system as its discrimination threshold changes. The curve is created by plotting the true positive rate against the false positive rate under different threshold settings. Here, the true positive rate is called sensitivity; the false positive rate is calculated as 1-specificity. Therefore, the ROC curve is a clinical method for selecting the optimal cutoff value based on a graphical display of the true positive rate (sensitivity) versus the false positive rate (1-specificity) within a range of cutoff values. Its accuracy is expressed as the area under the ROC curve (AUC), which provides a practical parameter for comparing test performance. In the ROC curve, the closer the AUC value is to 1, the more sensitive and specific the test is, while an AUC value close to 0.5 indicates that the test is neither sensitive nor specific.

[0029] When referring to the difference between a test sample and a control sample or a reference sample, the term "statistical difference" or "statistical significance" refers to the situation where the probability of the groups being identical is less than 5% (e.g., P < 0.05) when using an appropriate statistical analysis method, i.e., the probability of obtaining the same result on a completely random basis is less than 5 times in 100 attempts. Similarly, if the probability of the groups being identical is less than 1‰ (e.g., P < 0.001), i.e., the probability of obtaining the same result on a completely random basis is less than 1 time in 1000 attempts. In the present invention, we selected an appropriate statistical analysis method, for example, depending on whether the study variable is normally distributed (using the Kolmogorov-Smirnov test), we selected a parametric model test such as a t-test or an ANOVA test (normal distribution), or a non-parametric model such as a MannWhitney U test or a Kruskal-Wallis test (non-normal distribution). At the same time, the CD23 and PTX3 protein chip detection data were also double-logarithmic transformed (base 2) so that the CD23 and PTX3 protein detection data after log2 transformation conformed to the normal distribution (determined by Kolmogorov-Smirnov test).

[0030] The technical solutions in the embodiments of the present application are described clearly and completely below in conjunction with the accompanying drawings. The following examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental methods in which specific conditions are not specified in the examples are generally performed under conventional conditions or under conditions recommended by the manufacturer.

[0031] Example

[0032] S1 Clinicians first recruit patients who meet the ICD-10 / DSM-5 diagnostic criteria for depression and are currently in the acute phase of a first-episode or recurrent depressive episode, as well as healthy controls, for psychiatric and clinical assessments. Figure 1 shown.

[0033] In this embodiment, a total of 120 research subjects were included, including 30 healthy controls, 30 patients with anxious depression, 30 patients with atypical depression, and 30 patients with anxious depression.

[0034] In this example, the inclusion and exclusion criteria for healthy controls and patients with depression and its three different clinical subtypes (anxious depression, atypical depression, and anxious depression) were:

[0035] Healthy controls

[0036] Inclusion criteria:

[0037] (1) Age 18-55 years old;

[0038] (2) no family history of mental illness or mental illness;

[0039] (3) total score of HAMD-17 scale ≤ 5 points;

[0040] (4) No history of neurodegenerative diseases, brain trauma, or cerebrovascular disease;

[0041] (5) No substance abuse or addiction;

[0042] (6) No unstable angina, myocardial infarction, congestive heart failure, severe liver cirrhosis, acute or chronic renal failure, severe diabetes, aplastic anemia, epilepsy, or other serious heart, liver, kidney, endocrine, or blood system diseases or diseases that may interfere with the test evaluation (laboratory abnormality index is more than 2 times higher than the normal value);

[0043] (7) Voluntarily participate in the study and sign the informed consent form.

[0044] Exclusion criteria:

[0045] (1) Patients with a history of mental illness, severe physical illness, cerebrovascular disease, or brain trauma were excluded;

[0046] (2) Patients with a history of severe allergic reactions or immune system diseases were excluded;

[0047] (3) Use of anti-inflammatory drugs or immunosuppressants within 1 month;

[0048] (4) Patients with brain trauma or severe physical illness;

[0049] (5) Pregnant, breastfeeding women or those planning to become pregnant.

[0050] People with depression

[0051] Inclusion criteria:

[0052] (1) Patients who meet the diagnostic criteria for a depressive episode in ICD-10 / DSM-5 and are currently in the acute phase of a first or recurrent depressive episode;

[0053] (2) Age 18-55 years old;

[0054] (3) total score of 17 items of Hamilton Depression Rating Scale (HAMD-17) ≥17 points;

[0055] (4) had not taken any antidepressant drugs, physical therapy or psychological treatment within six months before enrollment;

[0056] (5) Have a certain level of education and be able to understand the research content;

[0057] (6) Volunteer to participate in this study and sign a written informed consent.

[0058] Exclusion criteria:

[0059] (1) Secondary depression caused by other organic diseases;

[0060] (2) secondary depression caused by medication;

[0061] (3) alcohol or other substance abuse and dependence;

[0062] (4) Pregnant, breastfeeding women or those planning pregnancy;

[0063] (5) serious suicide attempt (HAMD-17 item 3 “Suicide” score ≥ 3 points, or suicidal behavior during the current depressive episode);

[0064] (6) comorbidity with other mental disorders;

[0065] (7) Patients with serious physical illness.

[0066] Among them, the three clinical subtypes of depression patients (melancholic depression, atypical depression, and anxious depression) are mainly classified and determined based on the 30-item Depression Symptom Self-Rating Scale (IDS-30) and the HAMD-17 scale, as shown below:

[0067] (1) Melancholic depression

[0068] The assessment is mainly based on the scores of relevant items in IDS-30, which requires that:

[0069] ① A score of 2 or more on at least one of IDS-30 item 9 (mood changes) or item 21 (ability to experience pleasure or enjoyment (excluding sexual activity))

[0070] ② The presence of at least three of the following five characteristics: extreme despair or melancholy, or emotional emptiness, extremely obvious depression; depressive symptoms are worse in the morning; irritability; psychomotor agitation or retardation; severe anorexia or weight loss.

[0071] (2) Atypical depression

[0072] The judgment is mainly based on the relevant items in IDS-30, which require the following to be met at the same time:

[0073] ① Item 21 of IDS-30 ≥ 2 points;

[0074] ② Increased appetite or significant weight gain;

[0075] ③ Sensitive interpersonal relationships;

[0076] ④ Lead palsy (heaviness, or feeling of heaviness in the arms or legs).

[0077] (3) Anxiety-type depression

[0078] The determination of anxious depression is mainly based on the relevant items of the HAMD-17 scale, which requires the following to be met at the same time:

[0079] ① The anxiety / somatic factor score of the HAMD-17 scale (including items 10, 11, 12, 13, 15, and 17) is ≥ 7 points;

[0080] ② At least two of the following five items exist: feeling nervous or uneasy; feeling unusually uneasy; having trouble concentrating because of worry; fearing that something terrible will happen; and fearing losing control of oneself.

[0081] S2 collected plasma samples and used protein chip technology to measure the patient's plasma CD23 and PTX3 protein levels.

[0082] In this example, a quantitative protein chip kit was used to detect the expression levels of CD23 and PTX3 proteins in plasma samples.

[0083] The kit contains the following items: quantitative protein glass chip (QAH-CUSTOM), sample diluent, 20× wash solution I, 20× wash solution II, standard mixture, immunoglobulin G antibodies against CD23 and PTX3 proteins, Cy3-streptavidin, slide cleaning and drying device, sealing strips, etc.

[0084] The specific detection implementation method is as follows:

[0085] (1) Dry quantitative protein chip:

[0086] Take out the quantitative protein chip, equilibrate it at room temperature for 20-30 minutes, and then place the chip in a vacuum desiccator for 1-2 hours to completely dry the protein chip.

[0087] (2) Configuration standard:

[0088] ① Add 500 μL of sample diluent to the vial containing the cytokine standard mixture to reconstitute the standard. Before opening the vial, quickly centrifuge and gently pipette up and down to dissolve the powder. Label this vial Std 1.

[0089] ② Label 6 clean centrifuge tubes as Std2, Std3 to Std7, and add 200 μL of sample diluent to each tube.

[0090] ③ Take 100 μL of Std 1 and add it to Std 2 and mix gently. Then take 100 μL of Std 2 and add it to Std 3. Continue to dilute the mixture to Std 7 to obtain the standard solution.

[0091] (3) Draw 100 μL of the sample dilution into another new centrifuge tube and mark it as CT as a negative control.

[0092] (4) Remove the completely dried glass slide and add 100 μL of sample diluent to each well. Incubate on a shaker at room temperature for 1 hour to block the quantitative antibody chip. Then wash with buffer.

[0093] (5) Aspirate the buffer in each well, add 60 μL of standard solution and sample (sample diluted 2 times for loading) to the well, and incubate at 4°C overnight.

[0094] (6) Wash the slides using a chip washer. In this application, the Thermo Scientific Well WashVersa chip washer was used to wash the slides. The process is divided into two steps: first, wash with 1× Wash Solution I, 250 μL of 1× Wash Solution I per well, wash 10 times, shake for 10 seconds each time, select high shaking intensity, and dilute 20× Wash Solution I with deionized water. Then, switch to 1× Wash Solution II for washing, 250 μL of 1× Wash Solution II per well, wash 6 times, shake for 10 seconds each time, select high shaking intensity, and dilute 20× Wash Solution II with deionized water.

[0095] (7) Incubate with antibody mixture

[0096] Centrifuge the eppendorf tube containing the mixture of CD23 and PTX3 antibodies, then add 1.4 ml of sample diluent, mix well and centrifuge again quickly, then add 80 μL of CD23 and PTX3 antibodies to each well and incubate on a shaker at room temperature for 2 hours.

[0097] (8) Wash again, following the same steps as (6).

[0098] (9) Incubation with Cy3-streptavidin

[0099] Centrifuge the Cy3-streptavidin Eppendorf tube, then add 1.4 ml of sample diluent, mix thoroughly, and quickly centrifuge again. Add 80 μL of Cy3-streptavidin to each well, wrap the quantitative protein chip with aluminum foil, and incubate in the dark at room temperature on a shaker for 1 hour.

[0100] (10) Wash again, following the same steps as (6).

[0101] (11) Place the chip in a washer-dryer, centrifuge at 1000 rpm and dry thoroughly.

[0102] (12) Fluorescence detection was performed on the dried glass chip. In this example, an InnoScan 300 Microarray Scanner fluorescence scanner was used to scan the signal. The scanning parameters were: Wavelength: 532 nm; Resolution: 10 μm. The Cy3 or green channel (excitation frequency = 532 nm) was used, and the signal value was captured using Mapix software.

[0103] S3 uses QAH-CUST data analysis software to analyze data to obtain the expression levels of CD23 and PTX3 proteins in plasma samples. The details are as follows:

[0104] First, the original detection data of the protein chip experiment was normalized, that is, the raw data obtained by the chip scanning was subjected to chip background removal and the data of the standard product were averaged and normalized. Because the original measurement data was skewed, the original measurement data was normally distributed after double logarithmic transformation (log2) and then used for subsequent statistical analysis. In addition, the discrete degree of the data was analyzed, and data outside the mean ± 2 standard errors (SD) were not included in the data statistics. In the present embodiment, the number of discrete data (mean ± 2SD) of the healthy control population and the melancholic depression, atypical depression, and anxious depression groups were 1, 3, 3, and 2 respectively. The original measurement values ​​and discrete values ​​of CD23 and PTX3 proteins are specifically shown in Table 1 below.

[0105] Table 1 Original measured values ​​of plasma CD23 and PTX3 proteins and their double logarithmic transformed values

[0106]

[0107]

[0108]

[0109]

[0110] Note: Italic numbers are discrete values, defined as values ​​outside the mean ± 2SD.

[0111] S4 The statistical results of the present invention are as follows Figure 2 As shown in the figure, "**" indicates statistically significant differences in CD23 and PTX3 protein expression between the atypical depression group and healthy controls, as well as between the melancholic depression and anxious depression groups (P < 0.01). Therefore, plasma CD23 and PTX3 protein levels were significantly elevated in the atypical depression group, and the differences were statistically significant compared with healthy controls and the two most common clinical subtypes of depression, melancholic depression and anxious depression.

[0112] In addition, the data is further analyzed and the results of the ROC curve are as follows Figure 3-4 As shown in the data, the AUC values ​​of CD23 protein in patients with atypical depression were 0.791, 0.748 and 0.726 respectively compared with healthy controls and patients with melancholic depression and anxious depression, while the AUC values ​​of PTX3 protein in patients with healthy controls and patients with melancholic depression and anxious depression were 0.760, 0.843 and 0.777 respectively, indicating that CD23 and PTX3 can both be used as objective biomarkers for atypical depression and have extremely excellent diagnostic efficacy for subtype classification.

[0113] By detecting the levels of CD23 and PTX3 proteins in the present invention, patients with atypical depression can be accurately diagnosed, thereby avoiding the impact of misdiagnosis or missed diagnosis on the accuracy and pertinence of the treatment plan, and improving work efficiency.

[0114] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the description is to be regarded as illustrative rather than restrictive.

Claims

1. Use of a biomarker in the preparation of a product for screening atypical depression or for classifying and diagnosing atypical depression subtypes, characterized in that: The biomarkers are CD23 and PTX3 proteins.

2. Use of a reagent for detecting the content of a biomarker in preparing a kit for screening atypical depression or for classifying and diagnosing atypical depression subtypes, characterized in that: The biomarkers are CD23 and PTX3 proteins.

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