Application of reagent for detecting content or expression level of gene TNFR1 in sample in preparation of product for diagnosing and / or predicting individual epilepsy

Abstract

The invention discloses an application of TNFR1 (Tumor Necrosis Factor Receptor 1) as a marker in diagnosing and / or predicting epilepsy. The reagent for detecting the content or the expression level of the gene TNFR1 in the sample is applied to preparation of a product for diagnosing and / or predicting epilepsy; the sample type is selected from one or more of cerebrospinal fluid and serum. According to the application, it is found for the first time that TNFR1 in cerebrospinal fluid or serum can be used as a biomarker for diagnosing and / or predicting epilepsy, and whether a subject is about to attack, whether the subject suffers from epilepsy and how the severity of the epilepsy can be judged by detecting the content of TNFR1 in cerebrospinal fluid or serum; and the prognosis condition of the epilepsy patient, such as the epilepsy development speed and the response condition to the treatment, can also be judged.

Description

Technical Field

[0001] This application relates to the field of biomarkers, specifically to the application of reagents for detecting the content or expression level of the gene TNFR1 in a sample in the preparation of products for diagnosing and / or predicting individual epilepsy, wherein TNFR1 is used as a biomarker for diagnosing and / or predicting individual epilepsy. Background Technology

[0002] Epilepsy is a clinical syndrome characterized by sudden, abnormal electrical discharges in localized neurons of the brain, leading to transient brain dysfunction. The global prevalence of epilepsy is 0.5%–1%, with an annual mortality rate of up to 1 per 1,000. Drug therapy is currently the first-line treatment for epilepsy; however, over 30% of patients develop drug-resistant epilepsy due to ineffective drug control or intolerance of side effects. Frequent seizures and long-term use of antiepileptic drugs not only cause severe physical harm but also affect patients' social, psychological, and mental well-being. Early diagnosis of epilepsy allows patients to seek effective treatment as early as possible. Early diagnosis of refractory epilepsy relies heavily on early clinical characteristics, such as the frequency, duration, and type of seizures before initial treatment, as well as the response to the first-line antiepileptic drug. However, these diagnostic criteria lack accuracy and objectivity. Therefore, finding objective, accurate, and specific early warning and diagnostic methods is a crucial issue that urgently needs to be addressed.

[0003] The use of biomarkers facilitates rapid and effective disease diagnosis. In the field of bioassay, almost every type of disease has corresponding biomarkers, such as neurological diseases like Alzheimer's, metabolic diseases like diabetes, and infectious diseases. However, biomarkers that can effectively predict and diagnose epilepsy are still relatively rare.

[0004] TNFR1, also known as TNFRSF1A, CD120a, or p55, is a type I transmembrane glycoprotein with a molecular weight of 55 kDa, belonging to the tumor necrosis factor (TNF) receptor superfamily. In recent years, an increasing number of researchers have discovered that TNFR1 is widely involved in the pathophysiological processes of various diseases, such as inflammation, autoimmune diseases, tumors, diabetes, COVID-19 infection, and neurological diseases. Some studies suggest that TNFR1 could serve as a biomarker for the risk of non-relapse mortality in hematopoietic stem cells after transplantation, but its role as a biomarker for epilepsy has not been reported or studied. Summary of the Invention

[0005] Therefore, it is necessary to address the technical problem of the lack of biomarkers for rapid and accurate diagnosis of epilepsy by providing at least one biomarker, particularly a reagent for detecting the content of the gene TNFR1 or its expression level in a sample, in the preparation of products for diagnosing and / or predicting individual epilepsy.

[0006] A first aspect of this application provides the use of a reagent for detecting the content of the gene TNFR1 or its expression level in a sample in the preparation of products for diagnosing and / or predicting individual epilepsy; said sample is derived from one or more of cerebrospinal fluid and serum.

[0007] In some embodiments, the reagent is selected from one or more of the following: a specific probe of the TNFR1 gene, a gene chip, specific primers, and an anti-TNFR1 antibody.

[0008] In some embodiments, the reagent includes an anti-TNFR1 antibody, and the content or expression level of the TNFR1 gene in the detection sample includes: determining the expression level of the TNFR1 gene using an ELISA method.

[0009] In some embodiments, the reagents include specific primers and specific probes for the gene TNFR1, and the detection of the content or expression level of the gene TNFR1 in the sample includes: determining the expression level of the gene TNFR1 using real-time quantitative reverse transcription polymerase chain reaction.

[0010] In some embodiments, detecting the content or expression level of the TNFR1 gene in the sample includes comparing the content or expression level of the TNFR1 gene in the sample with a control.

[0011] In some implementations, the sample type is cerebrospinal fluid, and the content or expression level of the gene TNFR1 is higher than that of the negative control, indicating that the subject corresponding to the sample detected by the reagent has epilepsy, epilepsy with increased severity, epilepsy about to occur, or epilepsy is currently occurring; the negative control is the content or expression level of the gene TNFR1 in the cerebrospinal fluid of a non-epileptic subject.

[0012] In some implementations, the sample type is cerebrospinal fluid, and the content or expression level of the gene TNFR1 is lower than that of the positive control, indicating that the subject corresponding to the sample detected by the reagent does not have epilepsy or the symptoms are relieved; the positive control is the content or expression level of the gene TNFR1 in the cerebrospinal fluid of a subject who is known to have epilepsy.

[0013] In some embodiments, the sample type is serum, and the content or expression level of the gene TNFR1 is lower than that of the negative control, indicating that the subject corresponding to the sample detected by the reagent has epilepsy, epilepsy with increased severity, epilepsy about to occur, or epilepsy is currently occurring; the negative control is the content or expression level of the gene TNFR1 in the serum of non-epileptic subjects.

[0014] In some implementations, the sample type is serum, and the content or expression level of the gene TNFR1 is higher than that of the positive control, indicating that the subject corresponding to the sample detected by the reagent does not have epilepsy or the symptoms are relieved; the positive control is the content or expression level of the gene TNFR1 in the serum of a subject who is known to have epilepsy.

[0015] In some embodiments, the epilepsy is selected from the group consisting of refractory epilepsy, early-stage epilepsy, mid-stage epilepsy, and late-stage epilepsy.

[0016] In some embodiments, the epilepsy is selected from the group consisting of symptomatic focal epilepsy, symptomatic epilepsy, secondary epilepsy, childhood epilepsy, adolescent epilepsy, absence epilepsy, epilepsy syndrome, epileptiform seizures, and epilepsy caused by focal cortical dysplasia.

[0017] In some embodiments, the epileptic focus of the epilepsy is selected from a group consisting of the cortex, frontal lobe, temporal lobe, temporal insula, anterior temporal lobe, left cerebral hemisphere, right cerebral hemisphere, hippocampus, amygdala, and thalamus.

[0018] In some implementations, the individual is a mammal.

[0019] In some embodiments, the mammal is a human, pig, cow, sheep, dog, or rat.

[0020] A second aspect of this application provides a kit for diagnosing and / or predicting epilepsy in an individual, comprising reagents for detecting the content of the gene TNFR1 or its expression level in a sample; said sample is derived from one or more of cerebrospinal fluid and serum.

[0021] A third aspect of this application provides a system for diagnosing and / or predicting epilepsy in an individual, the system comprising:

[0022] The data processing module is used to calculate the TNFR1 gene content or expression level data received or input from the individual's cerebrospinal fluid or serum, and obtain the calculation results; and

[0023] The judgment and output module is used to judge whether the calculation result meets the preset judgment conditions in order to diagnose and / or predict individual epilepsy, and output the prediction result.

[0024] In some implementations, in the judgment and output module, when the calculation result meets the preset judgment condition, the output prediction result is "suffering from epilepsy, aggravated, about to have an epileptic seizure, or currently having an epileptic seizure"; when the calculation result does not meet the preset judgment condition, the output prediction result is "the subject does not have epilepsy or the symptoms have been relieved".

[0025] In some embodiments, the reference dataset includes the content or expression level of the gene TNFR1 in the cerebrospinal fluid or serum of multiple subjects with confirmed epilepsy and the content or expression level of the gene TNFR1 in the cerebrospinal fluid or serum of multiple non-epilepsy subjects.

[0026] In some implementations, the determination and output module includes a machine learning model that performs the calculations.

[0027] In some implementations, the machine learning model is selected from one or more of the generalized linear model (GLM), random forest (RF), and support vector machine (SVM).

[0028] A fourth aspect of this application provides a computer-readable storage medium storing a computer program that, when executed by a processor, can perform the functions of the system described in the third aspect.

[0029] A fifth aspect of this application provides a computer device including a memory and a processor, the memory storing a computer program and the processor executing the computer program to perform the functions of the system as described in the third aspect.

[0030] A sixth aspect of this application provides an apparatus for diagnosing and / or predicting epilepsy, comprising one or more of the system described in the third aspect, the computer-readable storage medium described in the fourth aspect, and the computer device described in the fifth aspect.

[0031] A seventh aspect of this application provides a method for diagnosing and / or predicting epilepsy, comprising:

[0032] Samples were extracted from the subjects, the samples being selected from one or more of cerebrospinal fluid and serum;

[0033] The content or expression level of the TNFR1 gene in the sample was determined;

[0034] The results of measuring the content or expression level of the TNFR1 gene in the sample were compared with one or more of the positive and negative controls.

[0035] An eighth aspect of this application provides a method for treating epilepsy, comprising: treating the subject after diagnosing and / or predicting the epilepsy using the method of the seventh aspect of this application.

[0036] This application is the first to discover that TNFR1 in cerebrospinal fluid and serum can serve as a biomarker for diagnosing and / or predicting epilepsy. It can be used for non-invasive diagnosis of epilepsy, prediction of seizure severity, and assessment of epilepsy remission. By detecting TNFR1 levels in cerebrospinal fluid or serum, it is possible to determine whether a subject is about to have a seizure, whether they have epilepsy, and the severity of the epilepsy. It can also assess the prognosis of epilepsy patients, such as the rate of epilepsy progression and response to treatment. The procedure is easy to perform, has a short duration, and possesses high sensitivity and specificity, making it of significant potential clinical value. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application. The various dimensions of each component shown in the drawings are arbitrarily shown; they may be precise or not drawn to scale. For example, to make the illustration clearer, the dimensions of some components are appropriately exaggerated in the drawings. Unless otherwise specified, the components in the drawings are not drawn to scale. The drawings of this application do not limit each dimension of each component.

[0038] Figure 1 The results of behavioral scores of rats in each group in one embodiment of this application are shown (Blank control: normal control group; Sham: sham-operated control group; Epilepsy: epilepsy model group).

[0039] Figure 2 The TNFRSF1A content in the brain tissue of rats in each group in one embodiment of this application (Sham: sham-operated control group; Epilepsy: epilepsy model group).

[0040] Figure 3 The TNFR1 content in brain tissue of each group in one embodiment of this application (Control: normal control group; Sham: sham-operated control group; Latencyperiod: pre-seizure period; Epilepsy: peak seizure period; Lateseizure period: post-seizure period).

[0041] Figure 4 The TNFR1 content in the serum of rats in each group in one embodiment of this application is shown (Blank control: normal control group; Sham: sham-operated control group; Epilepsy: epilepsy model group).

[0042] Figure 5 The TNFR1 content in the cerebrospinal fluid of rats in one embodiment of this application is shown (Blank control: normal control group, Sham: sham-operated control group; Epilepsy: epilepsy model group).

[0043] Figure 6 The TNFR1 content in the serum of clinical samples in one embodiment of this application (Healthy control: healthy volunteer group; Epilesy: epilepsy patient group; Seizure frequency: seizure frequency; Weekly: more than once a week; Monthly: once to three times a month; Annual: once to eleven times a year; Seizure free: epilepsy remission group; Seizure: epilepsy seizure group).

[0044] Figure 7 The ROC curves of TNFR1 in serum samples from clinical epilepsy patients and healthy controls in one embodiment of this application are shown (“Sensitivity” means sensitivity; “Specificity” means specificity).

[0045] Figure 8 The ROC curves of TNFR1 in epilepsy patients with high and low seizure frequency are shown in one embodiment of this application (“Sensitivity” means sensitivity; “Specificity” means specificity).

[0046] Figure 9 In one embodiment of this application (Healthy control: healthy volunteer group; Epilesy: epilepsy patient group; "Human serum TNFR1 concentration" refers to the concentration of TNFR1 in human serum; "Sensitivity" refers to sensitivity; "Specificity" refers to specificity).

[0047] Figure 10 The ROC curves of TNFR1 in epilepsy patients with high and low seizure frequency were used to verify the results in one embodiment of this application. (“Sensitivity” refers to sensitivity; “Specificity” refers to specificity.)

[0048] Figure 11 The TNFR1 content in the cerebrospinal fluid of clinical samples in one embodiment of this application (Non-epilepsy: non-epilepsy group; Epilepsy: epilepsy group). Detailed Implementation

[0049] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0051] The term "and / or" as used in this document encompasses any one of two or more of the related listed items, as well as any and all combinations of the related listed items. "Any and all combinations" includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, "A and / or B" includes three parallel options: A, B, and "a combination of A and B".

[0052] While existing technologies have revealed a correlation between TNFR1 expression levels in the hippocampus and epilepsy, the hippocampus, located between the thalamus and the medial temporal lobe, makes it impractical to diagnose epilepsy by detecting TNFR1 expression levels therein. This application creatively discovers a clear correlation between TNFR1 expression levels in cerebrospinal fluid and serum and epilepsy. The corresponding epilepsy types include symptomatic focal epilepsy, childhood epilepsy, and adolescent epilepsy, involving brain regions such as the frontal lobe, temporal lobe, temporal insula, and even the left or right hemispheres. It is not limited to epilepsy originating from the hippocampus but can reflect epilepsy originating from cortical, thalamic, or other points. Furthermore, both cerebrospinal fluid and blood are easily collected samples, thus possessing broader application and clinical translational value.

[0053] A first aspect of this application provides the use of a reagent for detecting the content of the gene TNFR1 or its expression level in a sample in the preparation of products for diagnosing and / or predicting individual epilepsy; said sample is derived from one or more of cerebrospinal fluid and serum.

[0054] Unless otherwise specified, the term "expression level" in this application is the same as "level" and refers to the absolute or relative amount of the biomarker TNFR1 in this application. The expression level of the biomarker TNFR1 in this application can be determined by various techniques. In particular, the absolute or relative amount of the biomarker TNFR1 in this application can be detected by using methods well known to those skilled in the art.

[0055] Unless otherwise specified, the term "serum" in this application refers to the pale yellow, transparent liquid separated from blood plasma after fibrinogen and certain clotting factors have been removed.

[0056] In some embodiments, the serum is obtained by: letting the blood sample stand at room temperature for 40 to 60 minutes, then centrifuging at 3000 to 3500 rpm for 10 to 15 minutes to separate the supernatant and obtain a serum sample.

[0057] Unless otherwise specified, the term "room temperature" in this application refers to a temperature range at an average indoor temperature, with an exemplary temperature range of 15 to 25°C.

[0058] In some embodiments, the reagent is selected from one or more of the following: a specific probe of the TNFR1 gene, a gene chip, specific primers, and an anti-TNFR1 antibody.

[0059] The reagents can be determined based on the method for detecting the expression level.

[0060] In one embodiment, Western blotting is used to detect the content or expression level of the gene TNFR1, and the reagent may include an anti-TNFR1 antibody.

[0061] Unless otherwise specified, the term "Western blotting" in this application refers to a method for detecting TNFR1 in a sample based on the specific binding of an antigen-antibody.

[0062] Unless otherwise specified, the term "antibody" in this application refers to a specific immunoglobulin targeting an antigenic site. The antibody in this application refers to an antibody that specifically binds to the TNFR1 protein described in this application, and can be manufactured according to conventional methods in the art. Antibodies can take the form of polyclonal or monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigen-binding site, and any other modified immunoglobulin molecule containing an antigen-binding site, provided that the antibody exhibits the desired biological binding activity.

[0063] In one embodiment, the expression level of the biomarker is detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). The reagent may be in the form of primers, probes, or a combination of primers and probes. RT-qPCR technology can quantify the mRNA level of a gene, offering short detection time, low cost, and high sensitivity and specificity. Unless otherwise specified, the term "RT-qPCR" (Reverse Transcription-Polymerase Chain Reaction) in this application refers to a technique that combines reverse transcription (RT) of RNA with polymerase chain amplification (PCR) of cDNA.

[0064] Unless otherwise specified, the term "primer" in this application refers to a nucleic acid sequence capable of forming a base pair complementary to the template strand and serving as a starting point for template strand replication; for example, its length may be 7-50 bases. Primers are usually synthesized, but naturally occurring nucleic acids may also be used. The primer sequence does not necessarily need to be completely identical to the template sequence, as long as it is sufficiently complementary to the template and can hybridize.

[0065] Unless otherwise specified, the term "probe" in this application refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few to hundreds of bases in length, which can specifically bind to mRNA and can determine the presence of a specific mRNA through labeling. Probes can be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, and RNA probes.

[0066] It should be noted that other technical means can also be used to detect the expression level of biomarkers, such as RNA sequencing, immunohistochemistry, flow cytometry, and in situ hybridization.

[0067] Unless otherwise specified, the term "RNA sequencing" in this application refers to the determination of the base sequence of RNA using sequencing technologies (such as single-molecule sequencing technology).

[0068] In one embodiment, detecting the content or expression level of the TNFR1 gene in the sample includes comparing the content or expression level of the TNFR1 gene in the sample with a control.

[0069] Unrestricted by any theory, it is believed that the content or expression level of the TNFR1 gene in cerebrospinal fluid is positively correlated with epilepsy.

[0070] In some embodiments, the sample type is cerebrospinal fluid. When the level of TNFR1 or its expression level in the cerebrospinal fluid of a subject is lower than that of a positive control, the subject corresponding to the sample tested by the reagent is considered not to have epilepsy or to have alleviated symptoms. The positive control is the level of the TNFR1 gene or its expression level in the cerebrospinal fluid of a subject who has a confirmed history of epilepsy.

[0071] In some embodiments, the sample type is cerebrospinal fluid. When the level of TNFR1 or its expression level in the cerebrospinal fluid obtained from the subject is higher than that in the negative control, the subject corresponding to the sample detected by the reagent is identified as having epilepsy, epilepsy with increased severity, epilepsy about to occur, or epilepsy currently occurring; the negative control is the level of the TNFR1 gene or its expression level in the cerebrospinal fluid of a non-epileptic subject.

[0072] In some embodiments, the sample type is serum. When the level of TNFR1 or its expression level in the serum of a subject is lower than that of a negative control, the subject corresponding to the sample detected by the reagent is considered to have epilepsy, epilepsy with increased severity, impending epileptic seizure, or currently experiencing an epileptic seizure. The negative control is the level of the TNFR1 gene or its expression level in the serum of a non-epileptic subject.

[0073] In some embodiments, the sample type is serum. When the level of TNFR1 or its expression level in the serum of a subject is higher than that in a positive control, the subject corresponding to the sample detected by the reagent is considered not to have epilepsy or to have relieved symptoms; the positive control is the level of the TNFR1 gene or its expression level in the serum of a subject who has been diagnosed with epilepsy.

[0074] Unless otherwise specified, “determination” includes a confirmed diagnosis, or a presumptive determination of a certain outcome or the probability of a certain outcome.

[0075] The epilepsy described in this application encompasses various types of epilepsy. The epilepsy may be one or more of the following: refractory epilepsy, early-stage epilepsy, mid-stage epilepsy, late-stage epilepsy, symptomatic focal epilepsy, symptomatic epilepsy, secondary epilepsy, childhood epilepsy, adolescent epilepsy, absence epilepsy, epilepsy syndrome, epileptiform seizures, and epilepsy caused by focal cortical dysplasia.

[0076] In some embodiments, the epileptic focus of the epilepsy is selected from a group consisting of the cortex, frontal lobe, temporal lobe, temporal insula, anterior temporal lobe, left cerebral hemisphere, right cerebral hemisphere, hippocampus, amygdala, and thalamus.

[0077] In some implementations, the individual is a mammal.

[0078] In some embodiments, the mammal is a human, pig, cow, sheep, dog, or rat.

[0079] A second aspect of this application provides a kit for diagnosing and / or predicting epilepsy in an individual, comprising reagents for detecting the content of the gene TNFR1 or its expression level in a sample; said sample is derived from one or more of cerebrospinal fluid and serum.

[0080] Unless otherwise specified, the term "diagnosis" in this application refers to the discovery, judgment, or recognition of an individual's health status or condition based on one or more symptoms, data, or other information relating to that individual. An individual's health status may be diagnosed as healthy / normal (e.g., not having epilepsy), or as unhealthy / abnormal (e.g., having epilepsy), or as having a specific degree of severity or progression of the disease (e.g., impending seizure, ongoing seizure). The term diagnosis includes the early detection of a disease / symptom associated with a specific illness or condition; the characteristics or classification of the disease; the discovery of the progression, cure, or recurrence of the disease; and the discovery of the individual's response to the disease after treatment or intervention.

[0081] In some embodiments, the kit contains a specific probe for the gene TNFR1.

[0082] In some embodiments, the kit contains specific primers for the TNFR1 gene.

[0083] In some embodiments, the kit contains specific primers and specific probes for the TNFR1 gene.

[0084] In some embodiments, the kit contains an anti-TNFR1 antibody.

[0085] In some embodiments, the kit is an ELISA kit and contains an anti-TNFR1 antibody.

[0086] In some embodiments, the kit includes a gene chip.

[0087] A third aspect of this application provides a system for diagnosing and / or predicting epilepsy in an individual, the system comprising:

[0088] The data processing module is used to calculate the TNFR1 gene content or expression level data received or input from the individual's cerebrospinal fluid or serum, and obtain the calculation results; and

[0089] The judgment and output module is used to judge whether the calculation result meets the preset judgment conditions in order to diagnose and / or predict individual epilepsy, and output the prediction result.

[0090] In some implementations, in the judgment and output module, when the calculation result meets the preset judgment condition, the output prediction result is "the subject has epilepsy, the severity has worsened, an epileptic seizure is about to occur, or an epileptic seizure is currently occurring"; when the calculation result does not meet the preset judgment condition, the output prediction result is "the subject does not have epilepsy or the symptoms have been relieved".

[0091] In some implementations, the judgment criterion is that the content or expression level of the TNFR1 gene in the cerebrospinal fluid of the subject is higher than that of the negative control. When this judgment criterion is met, the output prediction result is that the subject has epilepsy, the severity of epilepsy has worsened, an impending epileptic seizure is about to occur, or an epileptic seizure is currently occurring. Here, the negative control refers to the content or expression level of the TNFR1 gene in the cerebrospinal fluid of a non-epileptic subject.

[0092] In some implementations, the judgment criterion is that the content or expression level of the TNFR1 gene in the cerebrospinal fluid of the subject is lower than that of the positive control. When this judgment criterion is met, the output prediction result is that the subject does not have epilepsy or the symptoms are relieved. The positive control is the content or expression level of the TNFR1 gene in the cerebrospinal fluid of a subject who has a confirmed diagnosis of epilepsy.

[0093] In some implementations, the judgment criterion is that the content or expression level of the TNFR1 gene in the serum of the subject is lower than that of the negative control. When this judgment criterion is met, the output prediction result is that the subject has epilepsy, the severity has worsened, an impending epileptic seizure is imminent, or an epileptic seizure is currently occurring. The negative control is the content or expression level of the TNFR1 gene in the serum of a non-epileptic subject.

[0094] In some implementations, the judgment condition is that the level of the TNFR1 gene or its expression level in the serum of the subject is higher than that of the positive control. When this judgment condition is met, the output prediction result is that the subject does not have epilepsy or that the symptoms are relieved. The positive control is the level of the TNFR1 gene or its expression level in the serum of a subject who has a confirmed diagnosis of epilepsy.

[0095] In some embodiments, the reference dataset includes the content or expression level of the gene TNFR1 in the cerebrospinal fluid or serum of multiple subjects with confirmed epilepsy and the content or expression level of the gene TNFR1 in the cerebrospinal fluid or serum of multiple non-epilepsy subjects.

[0096] In some implementations, the determination and output module includes a machine learning model that performs the calculations.

[0097] In some implementations, the machine learning model is selected from one or more of the generalized linear model (GLM), random forest (RF), and support vector machine (SVM).

[0098] A fourth aspect of this application provides a computer-readable storage medium storing a computer program that, when executed by a processor, can perform the functions of the system described in the third aspect.

[0099] A fifth aspect of this application provides a computer device including a memory and a processor, the memory storing a computer program and the processor executing the computer program to perform the functions of the system as described in the third aspect.

[0100] The computer device can be a terminal. The computer device includes a processor, memory, communication interface, display screen, and input devices connected via a system bus. The processor provides computing and control capabilities. The memory includes non-volatile storage media and internal memory. The non-volatile storage media stores the operating system and computer programs. The internal memory provides an environment for the operation of the operating system and computer programs in the non-volatile storage media. The communication interface of the computer device is used for wired or wireless communication with external terminals; wireless communication can be achieved through Wi-Fi, mobile cellular networks, NFC (Near Field Communication), or other technologies. When the computer program is executed by the processor, it implements a method for determining the battery performance of an energy storage system. The display screen of the computer device can be an LCD screen or an e-ink display screen. The input devices of the computer device can be a touch layer covering the display screen, buttons, a trackball, or a touchpad located on the casing of the computer device, or an external keyboard, touchpad, or mouse, etc.

[0101] Those skilled in the art will understand that all or part of the processes in the above-described method embodiments can be implemented by a computer program instructing related hardware. The computer program can be stored in a non-volatile computer-readable storage medium. When executed, the computer program can include the processes of the embodiments described above. Any references to memory, databases, or other media used in the embodiments provided in this application can include at least one of non-volatile and volatile memory. Non-volatile memory can include read-only memory (ROM), magnetic tape, floppy disk, flash memory, optical memory, high-density embedded non-volatile memory, resistive random access memory (ReRAM), magnetic random access memory (MRAM), ferroelectric random access memory (FRAM), phase change memory (PCM), graphene memory, etc. Volatile memory can include random access memory (RAM) or external cache memory, etc. By way of illustration and not limitation, RAM can take many forms, such as Static Random Access Memory (SRAM) or Dynamic Random Access Memory (DRAM). The databases involved in the embodiments provided in this application may include at least one type of relational database and non-relational database. Non-relational databases may include, but are not limited to, blockchain-based distributed databases. The processors involved in the embodiments provided in this application may be general-purpose processors, central processing units, graphics processing units, digital signal processors, programmable logic devices, quantum computing-based data processing logic devices, etc., and are not limited to these.

[0102] A sixth aspect of this application provides an apparatus for diagnosing and / or predicting epilepsy, comprising one or more of the system described in the third aspect, the computer-readable storage medium described in the fourth aspect, and the computer device described in the fifth aspect.

[0103] A seventh aspect of this application provides a method for diagnosing and / or predicting epilepsy, comprising:

[0104] Samples were extracted from the subjects, the samples being selected from one or more of cerebrospinal fluid and serum;

[0105] The content or expression level of the TNFR1 gene in the sample was determined;

[0106] The results of measuring the content or expression level of the TNFR1 gene in the sample were compared with one or more of the positive and negative controls.

[0107] As used herein, the terms "subject" and "patient" are used interchangeably and refer to animals, such as humans. The term "subject" also includes "non-human mammals," such as rats, mice, rabbits, sheep, cats, dogs, cattle, pigs, and non-human primates. In some embodiments, the subject is a human subject.

[0108] An eighth aspect of this application provides a method for treating epilepsy, comprising: treating the subject after diagnosing and / or predicting the epilepsy using the method of the seventh aspect of this application.

[0109] As used in this application, the term "treatment" refers to a therapeutic intervention aimed at reversing, alleviating, improving, suppressing, slowing, or stopping the progression or severity of a condition associated with a disease or symptom. The term "treatment" includes reducing or alleviating at least one side effect or symptom of a disease or symptom. Treatment is generally "effective" if it reduces one or more symptoms or clinical markers. Alternatively, treatment is "effective" if the progression of the disease is reduced or stopped; that is, "treatment" includes not only improvement of symptoms but also the cessation, or at least slowing, of the expected progression or worsening of symptoms in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, reduction of one or more symptoms, reduction of disease severity, stabilization (i.e., non-worsening) of the disease state, delay or slowing of disease progression, improvement or relief of the disease state, and relief (whether partial or complete), whether detectable or undetectable.

[0110] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0111] Adult SPF-grade SD rats were purchased from Vital Rivers, but may also be supplied by other qualified companies. All procedures were conducted in accordance with the protocol approved by the Animal Ethics Committee of Guangzhou National Laboratory (No. 2022041) and strictly followed the relevant guidelines of international laboratory animal protection certification and assessment organizations.

[0112] The TNFR1 kit was purchased from Huamei Biotechnology Co., Ltd., and can also be purchased from R&D Biotechnology Company. Clinical samples were reviewed for medical ethics and came from epilepsy patients, non-epilepsy patients, and healthy volunteers at Guangdong Provincial Hospital of Traditional Chinese Medicine, and could also be collected from other qualified hospitals.

[0113] Example 1: A rat model of temporal lobe epilepsy induced by lithium-pilocarpine (LI-PILO).

[0114] Healthy SPF-grade Sprague-Dawley rats, 250-350g, were provided by Vital River Pharmaceuticals. They were divided into an epilepsy model group (n=10) and a normal control group (n=3). In the epilepsy group, rats were injected intraperitoneally with lithium chloride (127.2 mg / kg). Eighteen hours later, they were injected with scopolamine (1 mg / kg, intraperitoneal injection (ip)). Thirty minutes later, they were injected intraperitoneally with pilocarpine (30 mg / kg) to induce status epilepticus. Rats with a Racine behavioral score of 4 or higher and a seizure duration of more than 90 minutes were included in the study, and their seizures were terminated with diazepam (10 mg / kg, ip). Behavioral monitoring was conducted two months later to confirm the successful establishment of the epileptic seizure and chronic temporal lobe epilepsy model. Hippocampal tissue was harvested from the three rats with successful temporal lobe epilepsy models and flash-frozen in liquid nitrogen at -80℃ for later use.

[0115] Example 2: Construction of an animal model of cortical epilepsy and electroencephalographic-behavioral testing

[0116] Healthy SPF-grade Sprague-Dawley rats, 250-350g, were provided by Vital Rivers. They were divided into an epilepsy model group (n=15), a sham-operated control group (n=6), and a normal control group (n=6). After anesthesia, the rats were transferred to a stereotaxic apparatus. The heads were disinfected, the skin was incised, and the skull was exposed. Based on Paxions and Watson's *Stereotaxic Atlas of the Rat Brain*, a 1.0mm diameter, 1.5mm long cobalt wire was implanted into the left cerebral cortex of the epilepsy group (AP+2.0mm, ML+2.5mm), while a steel wire of the same diameter and length was implanted into the sham-operated control group. Bilateral motor cortex screw electrodes (AP+3.5mm, ML±2.5mm, DV-1.0mm) were implanted in both the epilepsy and sham-operated groups. Two 1.0mm diameter stainless steel screw electrodes were implanted into the left and right motor cortexes to record bilateral cortical EEG. A reference electrode was implanted in the occipital bone. Finally, the electrode interfaces were fixed to the skull surface with dental cement, and the skin was sutured. After the animals recovered from anesthesia, they were connected to an EEG machine for behavioral-EEG-video monitoring. The behavioral grading of epileptic seizures was based on the Racine criteria, with Grade 1: facial twitching and blinking; Grade 2: head nodding; Grade 3: unilateral limb twitching; Grade 4: bilateral limb twitching or standing; Grade 5: falling to the ground after seizures. The control group did not undergo any surgical procedures.

[0117] See results Figure 1 The results showed that no epileptic seizures were detected in the normal control group and the sham-operated group, while the epilepsy group exhibited significant epileptic seizures. The total number of seizures, total Racine score, and seizure duration were all significantly higher than those in the control group, as shown in the figure, indicating that the epilepsy model was successfully established. The successfully modeled rats were then subjected to tissue sampling. A portion of the cortical tissue was fixed with PFA, and another portion was flash-frozen in liquid nitrogen at -80℃ for later use.

[0118] mRNA transcriptome sequencing was performed on the lesion side of the epilepsy group and the control group in Examples 1 and 2 above, and the results of TNFR1 mRNA expression were extracted and analyzed.

[0119] See results Figure 2 The expression level of TNFR1 mRNA in the hippocampus of rats with temporal lobe epilepsy was significantly increased. The expression level of TNFR1 mRNA was also significantly increased in a rat model of cortical epilepsy, and the difference was even more significant compared to the control group.

[0120] Example 3: Immunohistochemical determination of TNFR1 distribution in cortical tissue

[0121] The PFA-fixed cortical tissue sections from Example 2 were subjected to immunohistochemical procedures such as dewaxing to water. The primary antibody was goat anti-rabbit TNFR1 antibody, and the secondary antibody was HRP. Both antibodies were purchased from Abcam. DAB was used for staining, and the tissues were observed and photographed under a microscope for comparison.

[0122] See results Figure 3 In the control group and sham-operated group (i.e., without seizures), TNFR1 was mainly expressed intracellularly. However, when a seizure was about to occur, the amount of TNFR1 secreted into the intercellular space increased significantly. During the peak of a seizure, a large amount of TNFR1 was secreted into the intercellular space. In the later stages of a seizure, when the frequency of seizures decreased, the amount of TNFR1 in the intercellular space began to decrease, but it was still higher than in the control group. This demonstrates that TNFR1 secretion is closely related to the level of seizures.

[0123] Example 4: Detection of TNFR1 levels in cerebrospinal fluid and blood samples

[0124] During the epileptic seizure period in rats (on the ninth day after surgery), cerebrospinal fluid and blood samples were collected from the three groups of rats in Example 2. The cerebrospinal fluid samples were aliquoted and stored at -80℃ for later use. The blood samples were allowed to stand at room temperature for 40-60 minutes, then centrifuged at 3000-3500 rpm for 10-15 minutes. The supernatant was separated to obtain serum samples, which were aliquoted and stored at -80℃ for later use.

[0125] The levels of TNFR1 in serum and cerebrospinal fluid were determined using an ELISA method. The kit was purchased from Huamei Biotechnology Co., Ltd., and the rat species was used. The procedures were strictly followed according to the kit instructions. A standard curve was plotted using CurveExpert 1.3 software, and the concentrations of the test samples were calculated, expressed in pg / ml. Each test sample and standard was replicated.

[0126] The results show:

[0127] There was no significant difference in serum TNFR1 levels between the sham-operated group and the normal group. However, the serum TNFR1 levels were significantly lower in the epilepsy group compared to the sham-operated group. Analysis of different seizure severity levels in the epilepsy group showed that rats with more than 20 seizures had significantly lower serum TNFR1 levels than rats with fewer than 20 seizures (see...). Figure 4 ).

[0128] TNFR1 levels in cerebrospinal fluid of rats in each group: Compared with the sham-operated group and the normal control group, the TNFR1 level in cerebrospinal fluid of the epilepsy group was significantly increased, while there was no significant difference between the sham-operated group and the normal control group (see...). Figure 5 ).

[0129] Example 5: Collection of clinical samples and detection of TNFR1 levels in serum and cerebrospinal fluid.

[0130] Clinical samples meeting ethical standards were obtained from Guangdong Provincial Hospital of Traditional Chinese Medicine. Cerebrospinal fluid (CSF) samples were collected from epilepsy patients in the case group, while the CSF sample control group consisted of non-epilepsy patients with neurological disorders requiring resection or lumbar puncture. Blood samples were collected from epilepsy patients in the case group, while the blood sample control group consisted of healthy individuals from a health checkup center.

[0131] First, cerebrospinal fluid (CSF) samples were collected from 13 cases (case group) and 8 cases (control group). Serum samples were collected from 82 cases and 55 cases (control group). The groups included symptomatic focal epilepsy, symptomatic epilepsy, secondary epilepsy, childhood epilepsy, adolescent epilepsy, absence epilepsy, epilepsy syndromes, epileptiform seizures, and epilepsy caused by focal cortical dysplasia. Epilepsy foci included the cortex, frontal lobe, temporal lobe, temporal insula, anterior temporal lobe, left cerebral hemisphere, right cerebral hemisphere, hippocampus, amygdala, and thalamus. The levels of TNFR1 in serum and CSF were measured using an ELISA kit purchased from Huamei Biotechnology Co., Ltd., and the species was human. ROC (Receiver Operating Characteristic Curve) analysis was also performed.

[0132] Subsequently, serum samples were collected from 45 epilepsy cases and 27 healthy controls to verify whether TNFR1 could serve as a serum marker for predicting the onset and severity of epilepsy.

[0133] Data analysis was performed using SPSS 19.0 statistical software. Quantitative data were expressed as mean ± standard deviation (x ± s), and P < 0.05 was considered statistically significant.

[0134] The results show:

[0135] Compared with healthy volunteers, patients with epilepsy had significantly lower serum TNFR1 levels. In patients with epilepsy, the higher the seizure frequency, the lower the TNFR1 level. Patients with seizure remission had significantly higher serum TNFR1 levels than patients with seizures (see...). Figure 6 ).

[0136] ROC curve of TNFR1

[0137] Figure 7 The ROC curves of TNFR1 in serum samples from clinical epilepsy patients and healthy controls showed an AUC (Area Under the Curve) of 0.7774, indicating that this indicator can effectively separate epilepsy patients from healthy individuals.

[0138] Figure 8The results showed that the ROC curve of TNFR1 in serum samples from patients with high seizure frequency (every week) and low seizure frequency (once a year to three times a month) had an AUC of 0.7517, indicating that this index can effectively separate patients with high seizure frequency from those with low seizure frequency.

[0139] Validation of TNFR1 biomarkers

[0140] Figure 9 The results showed that the ROC curves of TNFR1 in serum samples from clinical epilepsy patients and healthy controls were validated by collecting clinical samples. The AUC was 0.8937, the sensitivity was 81.48%, and the specificity was 86.67%.

[0141] Figure 10 The results showed that the ROC curves of TNFR1 in serum samples from patients with high seizure frequency (every week) and low seizure frequency (once a year to three times a month) had an AUC of 0.82, a sensitivity of 88.89%, and a specificity of 73.33%.

[0142] Figure 11 The results showed that the TNFR1 content in the cerebrospinal fluid of patients with epilepsy was significantly higher than that of patients without epilepsy.

[0143] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0144] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. The use of reagents for detecting the content or expression level of the gene TNFR1 in a sample in the preparation of products for diagnosing and / or predicting individual epilepsy; said sample is derived from one or more of cerebrospinal fluid and serum.

2. The application as described in claim 1, characterized in that, The reagents are selected from one or more of the following: specific probes for the TNFR1 gene, gene chips, specific primers, and anti-TNFR1 antibodies.

3. The application as described in claim 2, characterized in that, The reagent includes an anti-TNFR1 antibody, and the content or expression level of the TNFR1 gene in the detection sample includes: measuring the expression level of the TNFR1 gene using an ELISA method.

4. The application as described in any one of claims 1-3, characterized in that, The reagents include specific primers and specific probes for the gene TNFR1. The content or expression level of the gene TNFR1 in the detection sample includes: determining the expression level of the gene TNFR1 using real-time quantitative reverse transcription polymerase chain reaction.

5. The application as described in any one of claims 1-4, characterized in that, The detection of the content or expression level of the TNFR1 gene in the sample includes comparing the content or expression level of the TNFR1 gene in the sample with that of the control.

6. The application as described in claim 5, characterized in that, The content or expression level of the TNFR1 gene in the test sample meets one or more of the following criteria: The sample type is cerebrospinal fluid. If the content or expression level of the gene TNFR1 is higher than that of the negative control, the subject corresponding to the sample detected by the reagent is identified as having epilepsy, having a worsening of epilepsy, being about to have an epileptic seizure, or having an epileptic seizure. The negative control is the content or expression level of the gene TNFR1 in the cerebrospinal fluid of a non-epileptic subject. The sample type is cerebrospinal fluid. If the content or expression level of the gene TNFR1 is lower than that of the positive control, it is determined that the subject corresponding to the sample detected by the reagent does not have epilepsy or the symptoms are relieved. The positive control is the content or expression level of the gene TNFR1 in the cerebrospinal fluid of a subject who has been diagnosed with epilepsy. The sample type is serum. If the content or expression level of the gene TNFR1 is lower than that of the negative control, the subject corresponding to the sample detected by the reagent is identified as having epilepsy, having a worsening of epilepsy, being about to have an epileptic seizure, or having an epileptic seizure. The negative control is the content or expression level of the gene TNFR1 in the serum of non-epileptic subjects. The sample type is serum. If the content or expression level of the gene TNFR1 is higher than that of the positive control, it is determined that the subject corresponding to the sample detected by the reagent does not have epilepsy or the symptoms are relieved. The positive control is the content or expression level of the gene TNFR1 in the serum of a subject who has been diagnosed with epilepsy.

7. The application as described in any one of claims 1-6, characterized in that, The application satisfies one or more of the following: The epilepsy is selected from the group consisting of refractory epilepsy, early-stage epilepsy, mid-stage epilepsy, and late-stage epilepsy. The epilepsy mentioned is selected from the group consisting of symptomatic focal epilepsy, symptomatic epilepsy, secondary epilepsy, childhood epilepsy, adolescent epilepsy, absence epilepsy, epilepsy syndrome, epileptiform seizures, and epilepsy caused by focal cortical dysplasia. The epileptic focus of the epilepsy is selected from a group consisting of the cortex, frontal lobe, temporal lobe, temporal insula, anterior temporal lobe, left cerebral hemisphere, right cerebral hemisphere, hippocampus, amygdala, and thalamus. The individual in question is a mammal.

8. A kit for diagnosing and / or predicting epilepsy in an individual, characterized in that, It contains reagents for detecting the content of the gene TNFR1 or its expression level in a sample; the sample is derived from one or more of cerebrospinal fluid and serum.

9. A system for diagnosing and / or predicting epilepsy in an individual, characterized in that, The system includes: The data processing module is used to calculate the TNFR1 gene content or expression level data received or input from an individual's cerebrospinal fluid or serum, and obtain the calculation results; and The judgment and output module is used to judge whether the calculation result meets the preset judgment conditions in order to diagnose and / or predict individual epilepsy, and output the prediction result. Optionally, in the judgment and output module, when the calculation result meets the preset judgment condition, the output prediction result is "the subject has epilepsy, the severity has worsened, an epileptic seizure is about to occur, or an epileptic seizure is currently occurring"; when the calculation result does not meet the preset judgment condition, the output prediction result is "the subject does not have epilepsy or the symptoms have been relieved". Optionally, the reference dataset includes the content or expression level of the gene TNFR1 in the cerebrospinal fluid or serum of multiple subjects with confirmed epilepsy and the content or expression level of the gene TNFR1 in the cerebrospinal fluid or serum of multiple non-epilepsy subjects. Optionally, the judgment and output module includes a machine learning model for performing the calculation; Optionally, the machine learning model is selected from one or more of the generalized linear model (GLM), random forest (RF), and support vector machine (SVM).

10. A computer-readable storage medium storing a computer program, characterized in that, When the computer program is executed by the processor, it can perform the functions of the system as described in claim 9.

11. A computer device comprising a memory and a processor, the memory storing a computer program, characterized in that, The processor is used to execute the computer program to implement the functions of the system as described in claim 9.

12. An apparatus for diagnosing and / or predicting epilepsy, comprising one or more of the system of claim 9, the computer-readable storage medium of claim 10, and the computer device of claim 11.

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