Use of urine protein marker group in preparing a kit for predicting epileptic seizures

Abstract

The present invention relates to the field of epilepsy, and specifically to the use of a urine protein marker group in the preparation of a kit for predicting epileptic seizures, wherein the urine protein marker group includes at least one of antichymotrypsin, transthyretin, eukaryotic translation initiation factor 2α subunit and immunoglobulin γ-1 heavy chain constant region, and the kit includes a detection reagent for detecting the content of the urine protein marker group in a urine sample of a subject, and the subject is diagnosed as an epileptic patient and has a tendency to have an epileptic seizure. The urine protein marker group provided by the present invention can predict the risk of epileptic seizures in epileptic patients within 1 day, and provides epileptic patients with appropriate reaction and preparation time to take preventive measures or intervention measures in advance, which can enable epileptic patients to avoid other dangers associated with sudden and unexpected attacks of epilepsy, and avoid the psychological burden caused by a long prediction period.

Description

Technical Field

[0001] The present invention relates to the field of epilepsy, and in particular to use of a urine protein marker group in preparing a kit for predicting epileptic seizures. Background Art

[0002] Epilepsy is one of the most common chronic neurological diseases, affecting more than 70 million people worldwide. Epileptic seizures are caused by sudden, abnormal, self-sustaining electrical discharges in brain networks that usually last less than a few minutes. Symptoms of epileptic seizures may affect any part of the body and are usually manifested by the sudden recurrence of sensory disturbances, loss of consciousness, or convulsions.

[0003] Epilepsy kills approximately 120,000 people worldwide each year due to its sudden onset and lack of predictability. Unexpected seizures are often associated with unexpected deaths such as drowning, car accidents, and sudden unexpected death from epilepsy, which can lead to accidents, injuries, embarrassment, and expensive emergency medical care. Not knowing when a seizure will occur can lead to significant limitations on family, social, educational, and occupational activities, suggesting that the social consequences of epilepsy are often greater than the seizure itself. In addition to the serious injuries that can result from falls and other accidents during a seizure, the social stigma associated with epilepsy and its unpredictability can lead to severe self-destruction, irritability, and anxiety in people with epilepsy. This anxiety can lead to an increased incidence of seizures, and increased seizures can further increase chronic anxiety.

[0004] In summary, epileptic seizures are difficult to predict and are very dangerous. Predicting and warning of epileptic seizures in advance can improve the quality of life of epileptic patients and give them enough time to take action in advance. Existing technologies usually use electroencephalograms (EEGs) to analyze whether epileptic patients are in a seizure, or even predict seizures. However, this prediction is based on EEG data collected through long-term monitoring of brain signals of epileptic patients to predict imminent (e.g., within 1 hour) epileptic seizures. Therefore, this prediction requires a long time to continuously collect EEG data from epileptic patients, and does not allow epileptic patients to have enough time to take actions to control epileptic seizures (such as taking preventive medications) and arrange appropriate life plans in advance. Summary of the invention

[0005] The present invention provides a use of a urine protein marker group in preparing a kit for predicting epileptic seizures, characterized in that the urine protein marker group includes at least one of antichymotrypsin, transthyretin, eukaryotic translation initiation factor 2α subunit and immunoglobulin γ-1 heavy chain constant region, and the kit includes a detection reagent for detecting the content of the urine protein marker group in a urine sample of a subject, wherein the subject is diagnosed as an epileptic patient and has a tendency to epileptic seizures.

[0006] In some embodiments, the kit is used to predict the subject's risk of epileptic seizure within 1 day from the date of collection of the urine sample.

[0007] In some embodiments, the urine protein marker panel includes any two of antichymotrypsin, transthyretin, eukaryotic translation initiation factor 2 alpha subunit, and immunoglobulin gamma-1 heavy chain constant region.

[0008] In some embodiments, when the content of the urine protein marker group in the urine sample shows any of the following changes, the subject is predicted to have a positive risk of epileptic seizure: (a) increased antichymotrypsin, (b) increased thyroxine transporter protein, (c) increased immunoglobulin gamma-1 heavy chain constant region, (d) decreased eukaryotic translation initiation factor 2 alpha subunit.

[0009] In some embodiments, when the content of the urine protein marker group in the urine sample shows at least two of the following changes, the subject is predicted to have a strong positive risk of epileptic seizure: (a) increased antichymotrypsin, (b) increased thyroxine transporter protein, (c) increased immunoglobulin gamma-1 heavy chain constant region, (d) decreased eukaryotic translation initiation factor 2 alpha subunit.

[0010] In some embodiments, the subject is predicted to be seizure negative when the content of the urine protein marker group in the urine sample does not show the following changes: (a) increased antichymotrypsin, (b) increased thyroxine transporter protein, (c) increased immunoglobulin gamma-1 heavy chain constant region, (d) decreased eukaryotic translation initiation factor 2 alpha subunit.

[0011] In some embodiments, the subject is a patient with drug-resistant epilepsy.

[0012] In some embodiments, the subject has at least one epileptic seizure per month.

[0013] In some embodiments, the detection method includes one or more of mass spectrometry, immunochemical method, fluorescence analysis and radiochemical analysis.

[0014] In some embodiments, the subject is at least 18 years old.

[0015] In some embodiments, the epileptic seizure in the subject is focal in origin.

[0016] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects:

[0017] The existing technologies for predicting epileptic seizures usually rely on EEG data collected through long-term monitoring of brain signals of epileptic patients. In some cases, the continuous EEG data that needs to be analyzed may be as long as 24 hours, and different results may be interpreted due to different analysis methods (such as manual analysis and EEG-based machine learning model analysis). These existing technologies not only take up a lot of time of epileptic patients and the interpretation results are subjective, but also have poor predictive capabilities and cannot allow epileptic patients to have enough time (for example, only within a few hours) to take actions to prevent and control epileptic seizures and arrange appropriate plans.

[0018] The present invention creatively utilizes the changes in the content of a specific group of urine protein markers (i.e., antichymotrypsin, transthyretin, eukaryotic translation initiation factor 2α subunit, and immunoglobulin γ-1 heavy chain constant region) detected in epilepsy patients to predict the risk of epileptic seizures in epilepsy patients within 1 day (calculated from the date of urine sample collection). The present invention found that the above-mentioned urine protein markers not only showed obvious changes after the epileptic seizure, but actually changed 1 day before the epileptic seizure, earlier than other physiological indicators such as EEG. This may be because the brain of epileptic patients already has abnormal neuronal discharges and pathophysiological changes similar to clinical epileptic seizures 1 day before the epileptic seizure. Although the degree is not enough to cause clinical epileptic seizures, the neuroinflammation and immune response caused by it can be reflected in the changes in urine protein in epileptic patients. Therefore, the urine protein marker group provided by the present invention can make early and accurate predictions of epileptic seizures earlier than conventional physiological indicators such as EEG.

[0019] Compared with the prior art, the urine protein marker group provided by the present invention can better distinguish epileptic seizures (such as positive epileptic seizures, strongly positive epileptic seizures) from non-epileptic seizures (such as negative epileptic seizures), thereby predicting the risk of epileptic seizures in epilepsy patients within 1 day. The present invention provides epilepsy patients with appropriate reaction and preparation time (for example, a prediction period of 1 day) to take preventive measures or intervention measures in advance (for example, increasing the dosage of medication in advance, preventing epileptic seizures through vagus nerve stimulation or transcranial magnetic stimulation, avoiding the causes of epileptic seizures (such as staying up late, drinking) and other appropriate measures). The urine protein marker group provided by the present invention can not only enable epileptic patients to avoid sudden and unexpected epileptic seizures and other accompanying dangers to a certain extent, but also avoid the psychological burden caused by a long prediction period.

[0020] The present invention is based on the collection and detection of urine samples, which is non-invasive, convenient and can be obtained in large quantities. It is relatively easier to achieve standardization of detection, and while saving detection costs to a certain extent, it can still play a good early warning role. In addition, each urine protein marker in the urine protein marker group provided by the present invention, whether alone or in combination, can better predict epileptic seizures. Therefore, according to actual detection needs, a specific detection scheme (for example, detecting any one or two urine protein markers in the urine protein marker group of the present invention) can be selected to reduce detection costs and promote the popularization of epileptic seizure prediction. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.

[0022] Figure 1 The following are analysis results of differentially expressed proteins in accordance with an embodiment of the present invention (A is a graph showing differentially expressed proteins obtained by comparing urine samples of epilepsy patients 1 day before onset and 3 days before onset; B is a graph showing differentially expressed proteins obtained by comparing urine samples of epilepsy patients 0-2 hours after onset and 3 days before onset; C is a graph showing differentially expressed proteins obtained by comparing urine samples of epilepsy patients 1 day before onset and 3 days before onset, which overlap with differentially expressed proteins obtained by comparing urine samples of epilepsy patients 0-2 hours after onset and 3 days before onset);

[0023] Figure 2 This is a comparison chart of the changing trends of antichymotrypsin in urine samples of epilepsy patients and healthy controls;

[0024] Figure 3 This is a comparison chart of the changing trends of transthyretin in urine samples of epilepsy patients and healthy controls;

[0025] Figure 4 This is a comparison chart of the changing trends of eukaryotic translation initiation factor 2α subunit in urine samples of epilepsy patients and healthy controls;

[0026] Figure 5 This is a comparison chart of the changing trends of the constant region of the immunoglobulin gamma-1 heavy chain in urine samples of epilepsy patients and healthy controls;

[0027] Figure 6 A summary diagram of changes in urine protein markers provided by the present invention;

[0028] Figure 7This is a summary diagram of the baseline information of the epilepsy patients included in the present invention;

[0029] Figure 8 This is a result diagram of the main enriched pathways of the differentially expressed proteins screened in Example 3. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0031] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0032] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0033] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0034] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, even more typically + / - 0.5% of the stated value.

[0035] In this specification, some embodiments may be disclosed in a format that is in a certain range. It should be understood that this description of "being in a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0036] As used herein, the term "sample" or "specimen" refers to a biological material separated from a subject. The sample may comprise any biological material suitable for detecting the desired biomarker, and may comprise cellular material and / or non-cellular material from the subject. The sample may be separated from any suitable biological tissue or fluid, for example, whole blood, plasma, serum, skin, epidermal tissue, adipose tissue, liver tissue, urine or a cell sample. In some preferred embodiments, the sample is urine. The sample may be pretreated before actual detection, and the pretreatment methods include filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.

[0037] As used herein, the term "epilepsy" refers to the clinical phenomenon that an individual has two or more unprovoked epileptic seizures. Exemplary epileptic seizures include extremely brief loss of consciousness or muscle reflexes and severe and persistent convulsions. The frequency of epileptic seizures varies between different epilepsy patients. For example, some patients have a lower frequency, with less than one seizure per year; but some patients have a higher frequency, with multiple seizures per day. In this article, exemplary seizure types of epilepsy include generalized origin and focal origin seizures, and exemplary types of epilepsy include temporal lobe epilepsy, frontal lobe epilepsy, parietal lobe epilepsy, occipital lobe epilepsy, and epileptic encephalopathy, etc. In this article, the terms "epileptic seizure" and "epilepsy" are used interchangeably unless otherwise specified.

[0038] As used herein, the terms "subject", "individual" and "patient" are used interchangeably to refer to a mammal from which a sample is collected, unless otherwise indicated. A subject, individual or patient may be suffering from, at risk of or suspected of having a predisposition to a condition with epileptic seizures or symptoms of epileptic seizures. In some embodiments, typical subjects include humans who are susceptible to, suffering from or have had one or more epileptic seizures. In some preferred embodiments, the subject is an adult individual (i.e., the subject is 18 years of age and older).

[0039] As used herein, the term "proneness", e.g., "prone to having an epileptic seizure", refers to a reasonable medical probability that an event, such as an epileptic seizure, will occur or recur. The term "proneness" also includes the frequency with which such an event may occur before, after, or during continued treatment.

[0040] The "amount" or "level" of one or more biomarkers (referred to as "markers") refers to the absolute (quantitative) or relative (qualitative) amount or concentration of the biomarkers in a sample.

[0041] Any suitable method can be used to analyze the sample to determine the content of one or more markers in the sample. Exemplary suitable methods include chromatography (e.g., liquid chromatography (LC), high performance liquid chromatography (HPLC), gas chromatography (GC)), mass spectrometry (e.g., mass spectrometry (MS), tandem mass spectrometry (MS / MS2)), combined methods (gas chromatography / mass spectrometry (GC-MS), liquid chromatography / mass spectrometry (LC-MS), ultra-high performance liquid chromatography / tandem mass spectrometry (UHLC / MS / MS2) and gas-chromatography / tandem mass spectrometry (GC / MS / MS2)), immunochemical techniques such as enzyme-linked immunosorbent assay (ELISA) and combinations thereof, refractive index spectroscopy (RI), ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS). In addition, in some embodiments, the content of one or more markers can also be measured by indirect measurement, such as measuring the content of a certain compound (or multiple compounds) related to the content of the marker to be measured.

[0042] After determining the content of one or more markers in a sample obtained from a subject, the content is compared with a reference content to which it refers (e.g., a reference content derived from the subject himself and / or a selected reference content (e.g., the content of a reference sample selected according to conditions such as age, gender, epilepsy type, etc.)). The "reference content" of a marker can be an absolute or relative amount or concentration of a marker, the presence or absence of a marker, a range of amounts or concentrations of a marker, a minimum and / or maximum amount or concentration of a marker, an average amount or concentration of a marker, and / or a median amount or concentration of a marker. A suitable reference content of a marker can be determined by measuring the desired marker content of the same subject multiple times; it can also be determined by measuring the desired marker content of multiple specific subjects (e.g., subjects of the same gender and the same age) as a reference content for a specific population. The reference content and the marker content of a subject may vary depending on the specific technology used to measure the marker in a biological sample.

[0043] In some specific embodiments, for subjects whose epileptic seizure frequency is at least once a month on average, the reference content can be determined by regularly collecting samples (e.g., once a day) over a period of time (e.g., 2 weeks). If the subject has an epileptic seizure during the collection period, the content one week before the seizure can be regarded as the reference content.

[0044] The marker content of the subject can be compared with the reference content of the subject in a variety of ways, such as simple comparison, statistical analysis (e.g., t-test, Wilcoxon rank sum test). In some embodiments, the above comparison can be achieved by manual and / or automated systems. In some embodiments, the one or more marker content of the subject is higher than 5%, 10%, 15%, 20% or more of the reference content of the subject, which can be regarded as an increase in the one or more marker content of the subject. In some embodiments, the one or more marker content of the subject is lower than 5%, 10%, 15%, 20% or more of the reference content of the subject, which can be regarded as a decrease in the one or more marker content of the subject.

[0045] In some embodiments, when the content of the urine protein marker group in the urine sample of the subject does not show the following changes, the subject is predicted to be negative for epilepsy, that is, the subject has a low probability of epilepsy seizure within 1 day of collecting the urine sample (it can be regarded as no seizure and no preventive measures or intervention measures are needed in advance): (a) increased antichymotrypsin, (b) increased thyroxine transporter protein, (c) increased immunoglobulin gamma-1 heavy chain constant region, (d) decreased eukaryotic translation initiation factor 2α subunit.

[0046] In some embodiments, when the content of the urine protein marker group in the urine sample of the subject shows any of the following changes, the subject is predicted to have a positive risk of epileptic seizure, that is, the subject has a high probability of epileptic seizure within 1 day of collecting the urine sample, and the epilepsy patient should be advised to take preventive measures or intervention measures in advance: (a) increased antichymotrypsin, (b) increased thyroxine transporter protein, (c) increased immunoglobulin gamma-1 heavy chain constant region, (d) decreased eukaryotic translation initiation factor 2α subunit.

[0047] In some embodiments, when the content of the urine protein marker group in the urine sample of the subject shows at least two of the following changes, the subject is predicted to have a strong positive risk of epileptic seizure, that is, the probability of the subject having an epileptic seizure is extremely high within 1 day of collecting the urine sample, and epilepsy patients should be clearly advised to take preventive measures or intervention measures in advance to reduce the possible harm caused by epileptic seizures: (a) increased antichymosin, (b) increased thyroxine transporter protein, (c) increased immunoglobulin gamma-1 heavy chain constant region, (d) decreased eukaryotic translation initiation factor 2α subunit.

[0048] Embodiment 1

[0049] 1. Subjects

[0050] The present invention includes epilepsy patients who visited the Epilepsy Center of West China Hospital, Sichuan University from April 2022 to August 2022 (who were independently diagnosed with epilepsy by two physicians). The inclusion criteria for epilepsy patients are: (1) the frequency of epileptic seizures in the past 6 months is an average of 1 or more times per month; (2) the age is 18-50 years old. The exclusion criteria are: (1) patients with kidney disease, taking drugs that clearly affect changes in urine protein or renal function; (2) patients with abnormal urine routine, liver and kidney function test results; (3) patients with stroke, brain tumors, Alzheimer's disease, Parkinson's disease and other neurological diseases; (4) patients with other chronic diseases; (5) pregnant or lactating patients; (6) patients with no autonomy or other reasons unable to cooperate with the present invention. Healthy controls were matched with epilepsy patients in age and gender.

[0051] 2. Baseline Information

[0052] Baseline information collection was completed face-to-face between doctors and patients, including demographic information, epilepsy-related medical history information, examination results and medication history, and past medical history. Demographic information includes: gender, age, education level, and BMI. Epilepsy-related medical history information includes: age of epilepsy onset, course of disease, type of seizure, duration of seizure, seizure severity score, whether it is drug-resistant epilepsy, etc. Examination results include: cranial MRI, cranial PET-MRI, cranial CT, ordinary scalp EEG, video EEG, etc. Medication history includes: number of medications, type of medications, dosage of medications, etc. Past medical history includes: history of birth hypoxia, history of premature birth (gestational age <37 weeks), history of high fever convulsions, history of encephalitis and meningitis before onset, history of head trauma before onset, and family history of epilepsy.

[0053] Considering that the epileptic seizures of most epilepsy patients can be controlled after drug treatment, the present invention strictly selects drug-resistant epilepsy patients with an average seizure frequency of once a month or more for key research. The main baseline information is as follows: Figure 7 shown.

[0054] 3. Urine collection and diary recording

[0055] Urine collection was completed by the subjects at home. Each epilepsy patient collected urine continuously for 1-2 months, and each healthy control collected urine continuously for 3 days. The first urine on an empty stomach after getting up in the morning and the first urine within 1 hour after each epileptic seizure were collected. When collecting, use a disposable urine cup to collect midstream urine and pour it into a 15mL centrifuge tube, filling it to 12-13mL, and collect 2 tubes each time. After collection, a label with a serial number was attached to the centrifuge tube, and the date and time of urine collection were written on the tube cap. If there were multiple seizures in one day, the urine after the seizure was collected at most twice with an interval of more than 1 hour. Starting from the first day of urine collection, epilepsy patients recorded their diary every day in an electronic diary card. The record content included: (1) the date of urine collection; (2) the date and time of collection of morning urine every day; (3) the daily epileptic seizures and the date and time of urine collection after the seizure; (4) daily diet, exercise, sleep and menstrual volume for women.

[0056] 4. Urine transportation and storage

[0057] After each collection, the epilepsy patients immediately put the labeled centrifuge tubes containing urine into a -20℃ refrigerator. The epilepsy patients sent their urine to West China Hospital of Sichuan University on dry ice every 2 weeks. The urine of the epilepsy patients was received, sorted and registered by a dedicated person, and finally stored in the -80℃ refrigerator of the biological sample bank of West China Hospital of Sichuan University for future use.

[0058] Embodiment 2

[0059] 1. Urine pretreatment and labeling

[0060] Urine samples were inactivated and sterilized at 56°C for 30 min, and then 500 mL of urine samples were precipitated with cold acetone (urine:acetone = 1:4, v / v, -20°C) overnight. The precipitated urine samples were centrifuged at 3000 g for 5 min. The precipitate was resuspended in 200 mL of 8M urea containing 100 mM TEAB (tetraethylammonium bromide). The protein lysate was reduced and alkylated with 10 mMTCEP (tris(2-carboxyethyl)phosphine) and 40 mM IAA (iodoacetamide) at 30°C in the dark for 30 min. The solution was further diluted with 200 mL of 100 mM TEAB. Digestion was completed by digestion with an enzyme mixture of 5 mg trypsin and 1 mg Lys-C at 32°C for 12 h, and the reaction was terminated by adding 110 mL of 10% trifluoroacetic acid (TFA). Urine peptides were labeled with TMTpro16 plex reagent. Prior to TMTpro 16plex labeling, a batch design procedure was performed to minimize batch effects. The samples were divided into 27 batches for TMTpro 16plex labeling, and each batch contained the same number of samples, with samples from the same patient or control in the same batch. Mixed samples were included in each group as quality control.

[0061] 2. Mass spectrometry detection

[0062] The peptides from each batch were fractionated and analyzed separately with minor modifications. Briefly, TMT samples were chromatographed on a DIONEX UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) coupled to an XBridge PeptideBEH C18 column (300 A°, 5 mm, 4.6 mm*250 mm) (Waters). A gradient of 5% to 35% acetonitrile in 10 mM ammonia (pH = 10.0) was used at a flow rate of 1 mL / min to separate the samples. The peptides were separated into 60 fractions and combined into 30 fractions. The fractions were then dried and redissolved in mass spectrometry grade 2% acetonitrile / 0.1% formic acid. The redissolved peptides were analyzed in data-dependent acquisition (DDA) mode using the same U3000 HPLC system coupled to a Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Each fraction was loaded onto a precolumn (3 mm, 100 A°, 20 mm*75 mm) and then analyzed at a flow rate of 300 nL / min using a 60-min LC gradient (analytical column: 1.9 mm, 120 A°, 150 mm*75 mm; buffer A: 2% acetonitrile and 0.1% formic acid; buffer B: 98% acetonitrile and 0.1% formic acid). The buffer B gradient was uniformly changed from 5% to 28%. For mass spectrometry (MS) acquisition, the m / z range of MS1 was 350-1,800 Da, the resolution was 60,000, and the automatic gain control (AGC) target was set to 3e 6 , the maximum ion injection time was 50 ms. The first 15 precursor ions were selected for MS / MS experiments with a resolution of 45,000 and an AGC of 2e 5 The maximum ion injection time IT was 120 ms. The isolation window of the selected precursor was 0.7 m / z.

[0063] 3. Database search quantification and quality control

[0064] Proteomics data analysis FragPipe was used to search the MS raw data against a manually annotated and reviewed FASTA database of Homo sapiens proteins. Enzyme digestion was set to fully specific trypsin with two missed cleavages. Static modifications were set to TMTpro of lysine residues and N-terminal peptides and carbamidomethylation of cysteine. Variable modifications were set to oxidation of methionine and acetylation of N-terminal peptides. The precursor mass tolerance was set to 10 ppm and the product ion was 0.02 Da. A target false discovery rate (FDR) of 1% was allowed for peptide spectrum matching. The protein abundance ratio of the target sample to the mixed sample within each batch was used as the relative protein abundance for data analysis. To ensure the stability of the proteomics data, the coefficient of variation (CV) was used to determine the sample stability for the proteins identified in the mixed samples. The missing rate (NA ratio) was used to measure the missingness of the studied proteins. Proteins with a missing rate > 60% were removed, and the remaining missing data were filled with the KNN algorithm for subsequent analysis.

[0065] 4. Protein differential expression analysis

[0066] Samples from different time points of the same attack were paired. Principal component analysis (PCA) was performed first, and after removing outlier samples and paired samples, differentially expressed proteins (DEP) analysis between the two time points was performed. The normality test was performed on the differences of all protein data of paired samples. The average normality rate of the differences of all paired groups was 86.8%, so paired t-test was used for differentially expressed protein analysis. The differentially expressed protein analysis used the fold change (FC) and the test value P value to screen differentially expressed proteins (|log2FC|>0.15 and P value<0.05, FC is the protein value of the sample at the later time point / the protein value of the sample at the earlier time point).

[0067] 5. Pathway Analysis

[0068] The differentially expressed proteins were analyzed for pathways on Metascape (URL: https: / / metascape.org / gp / index.html# / main / step1). The pathway data sets analyzed included: Gene Ontology (GO) biological processes, Reactome gene data sets (Gene Sets), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

[0069] Embodiment 3

[0070] In this invention, a total of 19 epilepsy patients were analyzed (baseline information is shown in Figure 7) and 227 urine samples from 38 episodes of leukemia, as well as 66 urine samples from 22 healthy controls. After searching the library with FragPipe, a total of 4882 protein molecules were identified, and the median CV value of the mixed sample was 6%, indicating that the batch effect had little impact on sample detection. After eliminating protein molecules with more than 60% missing values, 3065 protein molecules remained.

[0071] Based on the above results, first, this example compares the urine samples of epilepsy patients 1 day before the onset (-1 day) with the urine samples 3 days before the onset (-3 days), and the number of differentially expressed proteins obtained is 227 ( Figure 1 A), the main enriched pathway is neutrophil degranulation. Next, this example compares the urine samples of epileptic patients 0 to 2 hours after the onset with the urine samples 3 days before the onset, and the number of differentially expressed proteins obtained is 525 ( Figure 1 B) The major enriched pathway is also neutrophil degranulation.

[0072] Then, this example further analyzed the differentially expressed proteins obtained by comparing 0 to 2 hours after the onset with 3 days before the onset, and the differentially expressed proteins obtained by comparing 1 day before the onset with 3 days before the onset. Figure 1 As shown in Figure C, there are 101 differentially expressed proteins with intersections, and all of them are rising (50) or falling (51). The above experimental results show that these proteins that change 0-2 hours after epileptic seizures actually have changed significantly 1 day before the onset of the seizure. The main enriched pathways are neutrophil degranulation, protein processing in the endoplasmic reticulum, and immunoglobulin production. Figure 8 These pathways are all related to inflammation and immune response.

[0073] Embodiment 4

[0074] Based on the results of Example 3, this example further screened and analyzed the differentially expressed proteins that were significantly changed (increased or decreased). The screening method of this example is as follows: 1. Select Figure 8 Proteins in the top three pathways (i.e., neutrophil degranulation, protein processing in the endoplasmic reticulum, and immunoglobulin production). Figure 8, the ordinate is the name of each pathway, and the abscissa is the value obtained by taking -log10 of the P value of the significance of the corresponding pathway. The larger the abscissa, the stronger the significance of the corresponding pathway. 2. Select proteins whose P values ​​1 day before the onset and 0-2 hours after the onset are both <0.1 and whose mean P values ​​rank in the top 3 (from small to large) in the above three pathways. 3. Select proteins that have no obvious changes in urine samples of normal people for three consecutive days. After screening, the following four proteins were finally screened in this embodiment: antichymotrypsin, thyroxine transporter, immunoglobulin gamma-1 heavy chain constant region, and eukaryotic translation initiation factor 2α subunit.

[0075] Figures 2 to 5 In the figure, the horizontal axis represents the time point of urine sample collection (in the figure of epileptic patients (i.e. Figures 2 to 5 The lower figure of the figure shows that the 3 days before the onset, 1 day before the onset, and 0-2 hours after the onset are respectively; in the figure of the healthy control (i.e. Figures 2 to 5 The upper figure shows the 1st, 2nd and 3rd day of the healthy control group for three consecutive days); the ordinate represents the relative protein abundance of the differentially expressed proteins detected (Log2 相对蛋白丰度 , i.e., the protein abundance ratio of the target sample to the mixed sample at a certain time point); each "point" in the figure represents the relative protein abundance detected in a sample at a certain time point, and the three points connected by a broken line represent the relative protein abundance of the same epileptic patient in the same seizure cycle or the same healthy control for three consecutive days.

[0076] Specifically, Figure 2 The figure below shows that compared with 3 days before the epileptic seizure, antichymotrypsin (Alpha-1-antichymotrypsin, UniProt accession number: P01011, SERPINA3 gene) in the urine samples of epileptic patients has an overall upward trend 1 day before the epileptic seizure, and further increases after the epileptic seizure. However, there is no uniform trend of antichymotrypsin in the urine samples of healthy controls ( Figure 2 ), which shows that antichymotrypsin does not change significantly in normal subjects.

[0077] like Figure 3 The figure below shows that compared with 3 days before the epileptic seizure, the thyroxine transport protein (Transthyretin, UniProt accession number: P02766, TTR gene) in the urine samples of epileptic patients has an overall upward trend 1 day before the epileptic seizure, and further increases after the epileptic seizure. However, there is no uniform trend of change in the thyroxine transport protein in the urine samples of healthy controls ( Figure 3 This indicates that transthyretin does not change significantly in normal people.

[0078] like Figure 4The figure below shows that compared with 3 days before the onset of epilepsy, the eukaryotic translation initiation factor 2 subunit 1 (Eukaryotic translation initiation factor 2 subunit 1, UniProt accession number: P05198, EIF2S1 gene) in the urine samples of epileptic patients has an overall downward trend 1 day before the onset of epilepsy, and further decreases after the onset of epilepsy. However, there is no uniform trend of change in the eukaryotic translation initiation factor 2 subunit 1 in the urine samples of healthy controls ( Figure 4 ), which indicates that the eukaryotic translation initiation factor 2α subunit has no obvious changes in normal people.

[0079] like Figure 5 The figure below shows that compared with 3 days before the epileptic seizure, the immunoglobulin gamma-1 heavy chain constant region (Immunoglobulin heavy constant gamma 1, UniProt accession number: P01857, IGHG1 gene) in the urine samples of epileptic patients has an overall upward trend 1 day before the epileptic seizure, and further increases after the epileptic seizure. However, there is no uniform trend of change in the immunoglobulin gamma-1 heavy chain constant region in the urine samples of healthy controls ( Figure 5 This indicates that the constant region of the immunoglobulin gamma-1 heavy chain does not change significantly in normal people.

[0080] In summary, this example found that the antichymotrypsin, transthyretin, eukaryotic translation initiation factor 2α subunit and immunoglobulin gamma-1 heavy chain constant region screened in this example changed significantly 1 day before and 0-2 hours after the onset of epilepsy in epileptic patients, but did not change significantly in normal people. In other words, antichymotrypsin, transthyretin, eukaryotic translation initiation factor 2α subunit and immunoglobulin gamma-1 heavy chain constant region can specifically distinguish epileptic patients who may be at risk of epileptic seizures from normal people (or epileptic patients who are negative for epileptic seizures), and can then be used as urine protein markers to predict epileptic seizures.

[0081] Embodiment 5

[0082] In combination with 16 actual epileptic seizure cases, this example further analyzed the four differentially expressed proteins screened out in Example 4.

[0083] First, in these 16 actual attack cases, at least one of the following changes was found in the urine samples: (a) increased antichymotrypsin, (b) increased transthyretin, (c) increased immunoglobulin gamma-1 heavy chain constant region, and (d) decreased eukaryotic translation initiation factor 2 alpha subunit.

[0084] Next, this example analyzes the distribution of the above four differentially expressed proteins in actual epileptic seizure cases. This example found that antichymotrypsin was elevated in 75.00% of actual epileptic seizure cases, transthyretin was elevated in 75.00% of actual epileptic seizure cases, eukaryotic translation initiation factor 2α subunit was reduced in 75% of actual epileptic seizure cases, and immunoglobulin γ-1 heavy chain constant region was elevated in 81.25% of actual epileptic seizure cases. The above results further verify that antichymotrypsin, transthyretin, eukaryotic translation initiation factor 2α subunit and immunoglobulin γ-1 heavy chain constant region can be used as urine protein markers for predicting epileptic seizures, and have high applicability. For example, when the detection cost is limited, any one of the above urine protein markers can be selected to predict the risk of epileptic seizures in epilepsy patients.

[0085] This example further evaluates the applicability of the above urine protein markers by combining the above urine protein markers in pairs. Figure 6 As shown, this embodiment found that when any two of the above-mentioned urine protein markers were selected to predict epileptic seizures, increased antichymotrypsin and / or increased transthyretin appeared in 93.75% of actual epileptic seizure cases (change 1), increased antichymotrypsin and / or decreased eukaryotic translation initiation factor 2α subunit appeared in 100.00% of actual epileptic seizure cases (change 2), increased antichymotrypsin and / or increased immunoglobulin γ-1 heavy chain constant region appeared in 93.75% of actual epileptic seizure cases (change 3), increased transthyretin and / or decreased eukaryotic translation initiation factor 2α subunit appeared in 87.50% of actual epileptic seizure cases (change 4), increased transthyretin and / or increased immunoglobulin γ-1 heavy chain constant region appeared in 93.75% of actual epileptic seizure cases (change 5), decreased eukaryotic translation initiation factor 2α subunit and / or increased immunoglobulin γ-1 heavy chain constant region appeared in 93.75% of actual epileptic seizure cases (change 6). And when the urine sample shows any of the following changes: increased antichymotrypsin and increased transthyretin, increased antichymotrypsin and decreased eukaryotic translation initiation factor 2α subunit, increased antichymotrypsin and increased immunoglobulin gamma-1 heavy chain constant region, increased transthyretin and decreased eukaryotic translation initiation factor 2α subunit, increased transthyretin and increased immunoglobulin gamma-1 heavy chain constant region, decreased eukaryotic translation initiation factor 2α subunit and increased immunoglobulin gamma-1 heavy chain constant region, the subject is more likely to have an epileptic seizure, and therefore has a higher risk of epileptic seizures. In other words, any two of the above urine protein markers can be selected to predict the risk of epileptic seizures in patients with epilepsy.

[0086] In summary, it has been verified that the urine protein markers in the urine protein marker group provided by the present invention, whether alone or in combination, can predict epileptic seizures within 1 day from the date of collection of the subject's urine sample and meet different detection needs.

[0087] Based on the role of the above-mentioned urine protein marker group in predicting epileptic seizures, in some embodiments of the present invention, when the content of the urine protein marker group in the urine sample of the subject does not show the following changes, the subject is predicted to be negative for epileptic seizures, that is, the probability of epileptic seizures in the subject is low within 1 day of collecting the urine sample (it can be regarded as no seizures, and no preventive measures or intervention measures need to be taken in advance): (a) increased antichymotrypsin, (b) increased thyroxine transporter protein, (c) increased immunoglobulin gamma-1 heavy chain constant region, (d) decreased eukaryotic translation initiation factor 2α subunit.

[0088] In some embodiments, when the content of the urine protein marker group in the urine sample of the subject shows any of the following changes, the subject is predicted to have a positive risk of epileptic seizure, that is, the subject has a high probability of epileptic seizure within 1 day of collecting the urine sample, and the epilepsy patient should be advised to take preventive measures or intervention measures in advance: (a) increased antichymotrypsin, (b) increased thyroxine transporter protein, (c) increased immunoglobulin gamma-1 heavy chain constant region, (d) decreased eukaryotic translation initiation factor 2α subunit.

[0089] In some embodiments, when the content of the urine protein marker group in the urine sample of the subject shows at least two of the following changes, the subject is predicted to have a strong positive risk of epileptic seizure, that is, the probability of the subject having an epileptic seizure is extremely high within 1 day of collecting the urine sample, and epilepsy patients should be clearly advised to take preventive measures or intervention measures in advance to reduce the possible harm caused by epileptic seizures: (a) increased antichymosin, (b) increased thyroxine transporter protein, (c) increased immunoglobulin gamma-1 heavy chain constant region, (d) decreased eukaryotic translation initiation factor 2α subunit.

[0090] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. Use of a urine protein marker group in preparing a kit for predicting epileptic seizures, characterized in that: The urine protein marker group includes antichymotrypsin, thyroxine transporter protein, eukaryotic translation initiation factor 2α subunit and immunoglobulin γ-1 heavy chain constant region. The kit includes a detection reagent for detecting the content of the urine protein marker group in a urine sample of a subject. The subject is diagnosed as an epileptic patient and has a tendency to have an epileptic seizure. The kit is used to predict the risk of epileptic seizure of the subject within 1 day from the date of collection of the urine sample. When the content of the urine protein marker group in the urine sample shows any of the following changes, the subject is predicted to have a positive risk of epileptic seizure: (a) increased antichymotrypsin, (b) increased thyroxine transporter protein, (c) increased immunoglobulin γ-1 heavy chain constant region, (d) decreased eukaryotic translation initiation factor 2α subunit.

2. The use according to claim 1, characterized in that When the content of the urine protein marker group in the urine sample shows at least two of the following changes, the subject is predicted to have a strong positive risk of epileptic seizures: (a) increased antichymotrypsin, (b) increased thyroxine transporter protein, (c) increased immunoglobulin gamma-1 heavy chain constant region, (d) decreased eukaryotic translation initiation factor 2 alpha subunit.

3. The use according to claim 1, characterized in that The subject is predicted to be negative for epilepsy seizures when none of the following changes are observed in the content of the urine protein marker group in the urine sample: (a) increased antichymotrypsin, (b) increased transthyretin, (c) increased immunoglobulin gamma-1 heavy chain constant region, and (d) decreased eukaryotic translation initiation factor 2 alpha subunit.

4. The use according to claim 1, characterized in that The subject is a patient with drug-resistant epilepsy.

5. The use according to claim 1, characterized in that The subject suffers from epileptic seizures at least once a month.

6. The use according to claim 1, characterized in that The detection method includes one or more of mass spectrometry, immunochemical method, fluorescence analysis and radiochemical analysis.

7. The use according to claim 1, characterized in that The subject is at least 18 years old.

8. The use according to claim 1, characterized in that The epileptic seizures in the subject are focal in origin.

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