Application of SERPING1 gene promoter methylation in predicting HBeAg seroconversion after treatment in patients with chronic hepatitis B virus and prediction system

Abstract

The application relates to the field of biological medicine, in particular to application of SERPING1 gene promoter methylation in predicting HBeAg seroconversion of a chronic hepatitis B patient after treatment and a prediction system. By detecting the SERPING1 gene promoter methylation rate in a to-be-detected sample, whether HBeAg seroconversion occurs in the patient after a certain period of treatment is predicted and judged. The application forms an in-vitro auxiliary prediction scheme capable of outputting a 96-week HBeAg seroconversion probability risk level, so as to assist in response probability evaluation and hierarchical management of an HBeAg-positive chronic hepatitis B patient receiving nucleos(t)ide analog treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application and prediction system of SERPING1 gene promoter methylation in predicting HBeAg seroconversion after treatment in patients with chronic hepatitis B. Background Technology

[0002] Chronic hepatitis B, caused by persistent hepatitis B virus infection, remains a significant public health issue. For HBeAg-positive chronic hepatitis B patients, HBeAg seroconversion, typically defined as the disappearance of HBeAg accompanied by the appearance of anti-HBe, is an important intermediate endpoint for evaluating the response to nucleoside (nucleotide) analogue antiviral therapy. Early achievement of this endpoint is generally associated with a better virological response and clinical outcome.

[0003] Nucleoside (acid) analogs such as entecavir and tenofovir disoproxil fumarate can effectively inhibit HBV replication, but HBeAg seroconversion is not simply a decrease in viral load; it also involves the host's immune status and its reconstitution. Therefore, even with similar treatment drugs and durations, the probability and timing of HBeAg seroconversion vary significantly among different patients within the same treatment cycle. Current clinical assessments typically refer to baseline HBeAg, HBsAg, HBV DNA, and alanine aminotransferase (ALT) levels. These indicators are easily affected by disease stage, viral load, and inflammatory activity, and a single indicator is insufficient to simultaneously characterize the host's immune regulatory status and its recovery potential.

[0004] To address the aforementioned differences, several technical approaches have been developed in related research and clinical practice: First, evaluating virological responses through long-term nucleoside (nucleotide) analogue therapy and dynamic monitoring of HBV DNA, HBeAg, and HBsAg; second, using baseline variables such as HBeAg, HBsAg, HBV DNA, and ALT for statistical regression, ROC curve, or nomogram analysis; third, screening for host immune-related candidate biomarkers using human peripheral blood mononuclear cells or peripheral blood transcriptomes; and fourth, quantitatively detecting DNA methylation at specific sites using techniques such as MSP (methylation-specific PCR) and Methylight (methylation-based quantitative PCR). Previous research in this application also explored candidate clues for the SERPING1 / C1-INH regulatory axis. SERPING1 (Serpin Family GMember 1) encodes C1 inhibitor (C1-INH), a key negative regulator of the classical complement pathway. It inhibits the activity of serine proteases C1r and C1s, as well as mannose-binding lectin-associated serine proteases (MASP), thereby preventing excessive complement activation and maintaining immune homeostasis. SERPING1 also participates in the regulation of the coagulation system and the kallikrein-kinin system; these known functions suggest its potential involvement in the coordinated regulation of innate immunity and inflammatory responses. The complement system is an important component of innate immunity, participating in pathogen recognition, inflammation regulation, and antiviral defense. Previous studies have indicated that SERPING1 expression is altered in various viral infections, but its application value in different stages of chronic HBV infection, HBeAg status changes, and nucleoside (nucleotide) analogue therapy responses still lacks standardized validation for specific clinical outcomes.

[0005] Based on the above research ideas, the main technical obstacles that are still difficult to solve are: (1) Virological indicators and liver injury indicators cannot directly cover the dimension of host immune regulation; (2) mRNA and protein are relatively volatile transient expression readings, and the public transcriptome is mostly used for candidate screening, which is difficult to directly convert into a unified individual detection threshold; (3) Although methylation detection can be quantitative, it is still necessary to clarify the specific promoter region, specific primers / probes after bisulfite conversion, internal reference and quality control, and form a reproducible calculation model with clinical variables in order to be used for clinical prediction; otherwise, the detection values ​​of different batches will be incomparable, and it will be difficult to convert the research correlation into a detection kit or prediction system; (4) Existing single technical routes can usually only give a certain detection value or statistical correlation, and cannot directly output the individualized conversion probability corresponding to the specified treatment cycle.

[0006] Based on this, this application proposes the application and predictive system of SERPING1 gene promoter methylation in predicting HBeAg serological conversion in patients with chronic hepatitis B. Using the SERPING1 / C1-INH axis as a candidate biological clue and examining SERPING1 promoter methylation, this study further explores a standardized closed loop for predicting the specific outcome of 96-week HBeAg serological conversion, from pre-treatment sampling and quality control to probability output. Summary of the Invention

[0007] This invention provides the application and prediction system of SERPING1 gene promoter methylation in predicting HBeAg seroconversion after treatment in patients with chronic hepatitis B. It forms an in vitro auxiliary prediction scheme that can output the risk level of HBeAg seroconversion probability at 96 weeks, in order to assist in the assessment of response probability and stratified management of HBeAg-positive chronic hepatitis B patients receiving nucleoside (acid) analogue therapy, and solves the problems existing in the prior art.

[0008] One of the technical solutions adopted in this invention is: This invention provides the application of reagents for detecting the DNA methylation level of the SERPING1 gene promoter in the preparation of products for predicting or assisting in the prediction of HBeAg serological conversion in HBeAg-positive chronic hepatitis B patients after treatment.

[0009] Furthermore, the assessment point for HBeAg serological seroconversion is 96 weeks after HBeAg-positive chronic hepatitis B patients receive nucleoside (acid) analogue therapy.

[0010] Furthermore, the reagents include primers, probes, or nucleic acid chips.

[0011] Furthermore, the reagent includes a primer pair and a probe for detecting the DNA methylation level of the SERPING1 gene promoter, wherein the upstream primer sequence of the primer pair is shown in SEQ ID NO.1, the downstream primer sequence of the primer pair is shown in SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO.3.

[0012] Furthermore, the methylation level of the SERPING1 gene promoter DNA can be detected by real-time quantitative methylight and expressed as the percentage of methylated reference (PMR).

[0013] Furthermore, the aforementioned primer pairs and probe sequences are as follows: SERPING1 methylation-specific upstream primer: 5'-TTTTAGTAGTTGGGATTATATGTGC-3' (SEQ ID NO. 1) SERPING1 methylation-specific downstream primer: 5'-ATATAAAACACCCAACTCAACA-3' (SEQ ID NO. 2) SERPING1 methylation-specific TaqMan probe: 5'-AAACTAACCGAACACGATAACTCAC-3' (SEQ ID NO. 3) Furthermore, in the above applications, a SERPING1 gene promoter DNA methylation level of <34.16% in the sample to be tested indicates a high probability or predicted conversion, while a level of ≥34.16% indicates a low probability or predicted no conversion.

[0014] Furthermore, the sample to be tested is a mononuclear cell in the peripheral blood of the subject; the methylation level of the SERPING1 gene promoter DNA in the sample is expressed as the methylation rate.

[0015] Furthermore, the product includes a kit or chip; the kit includes primer pairs and probes for detecting the methylation level of SERPING1 gene promoter DNA, genomic DNA extraction reagents, bisulfite modification reagents, methylation detection internal control primer pairs and probes, fully methylated positive control, unmethylated or template-free negative control, and real-time fluorescent PCR reaction reagents.

[0016] Furthermore, the sequences of the methylation detection internal reference primer pair and probe are as follows: ACTB methylation-specific upstream primer: 5'-TGGTGATGGAGGAGGTTTAGTAAGT-3' (SEQ ID NO.4) ACTB methylation-specific downstream primer: 5'-AACCAATAAAACCTACTCCTCCCTTAAA-3' (SEQ ID NO. 5) ACTB methylation-specific TaqMan probe: 5'-ACCACCACCCAACACACAATAACAAACACA-3' (SEQ ID NO.6) The TaqMan fluorescent probes for detecting the DNA methylation level of the SERPING1 gene promoter and the TaqMan fluorescent probes for methylation detection have a FAM fluorescent group at the 5' end and a BHQ1 quencher group at the 3' end.

[0017] The second technical solution adopted in this invention is: A predictive system is provided for predicting HBeAg seroconversion in HBeAg-positive chronic hepatitis B patients after nucleoside (nucleotide) analogue therapy. The evaluation time point for this predictive system is 96 weeks post-treatment. The predictive system includes: a sample detection unit, a data input unit, a data processing unit, and a result output unit. The sample detection unit obtains the baseline SERPING1 promoter methylation PMR. The data input unit receives the baseline SERPING1 promoter methylation PMR, baseline quantitative HBeAg, and alanine aminotransferase (ALT) results. The data processing unit calculates the probability of HBeAg seroconversion at 96 weeks after the model parameters are verified and locked using the original regression output. The result output unit outputs the probability value.

[0018] Furthermore, the sample detection unit uses the aforementioned reagents to detect baseline SERPING1 promoter methylation PMR.

[0019] Furthermore, the model of the data processing unit adopts Logistic multivariate regression and is converted into a nomogram. The model formula for calculating the probability is as follows: logit(P) = β0 + β1 PMR(SERPING1) + β2×ALT + β3×HBeAg, P=1 / [1+e^(-logit(P))], where P is the HBeAg serological conversion probability at 96 weeks, β0 to β3 are model parameters confirmed by the original regression output and locked before application, β0=2.450305819, β1=-0.097796161, β2=0.012203862, β3=-0.002170063, and e is the base of the natural logarithm ≈2.718281828.

[0020] Furthermore, in the calculation results of the above prediction system, when P≥0.320, it is determined to be a high probability or prediction conversion, and when P<0.320, it is determined to be a low probability or prediction not converted.

[0021] Furthermore, the prediction process using the aforementioned prediction system is as follows: S1: Obtain baseline clinical information and a prescribed volume of EDTA-anticoagulated peripheral blood before the subject begins prescribed nucleoside (acid) analogue therapy; S2: Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque and genomic DNA was extracted; S3: Perform bisulfite conversion on DNA, and evaluate the success of the conversion and amplification using specified internal controls and references; S4: Methylight reaction was performed using SERPING1 methylation-specific primers and TaqMan probes, with full methylation reference and internal reference used to calculate the SERPING1 promoter PMR; S5: Input the qualified PMR and baseline HBeAg and ALT into the following preset model formula: logit(P) = 2.450305819 - 0.097796161 × PMR + 0.012203862 × ALT -0.002170063 × HBeAg, P=1 / [1+e^(-logit(P))]; e is the base of the natural logarithm ≈2.718281828.

[0022] S6: Calculate and output the predicted probability of HBeAg serological conversion at week 96.

[0023] The beneficial effects of this invention are: 1. Compared with schemes that rely solely on conventional virology or liver function indicators, this invention introduces SERPING1 promoter DNA methylation, a relatively stable epigenetic marker, which can supplement HBeAg serological conversion prediction information from the perspective of host immune regulation; the sample source is peripheral blood mononuclear cells, which is minimally invasive and suitable for pre-treatment assessment.

[0024] 2. This invention studies the SERPING1 promoter DNA methylation level in samples from patients with chronic hepatitis B using designed specific primers and probes. The results show that the PMR of these biomarkers has effective predictive value for HBeAg seroconversion at 96 weeks after nucleoside (nucleotide) analogue treatment. Furthermore, by combining the PMR of these biomarkers with baseline HBeAg and ALT levels in the samples to establish a predictive model, it was found to exhibit good discrimination, calibration, and net clinical benefit in existing cohorts, demonstrating the feasibility of developing a software-based predictive system. Simultaneously, its application value as an effective predictive strategy was practically verified. Attached Figure Description

[0025] Figure 1 This is a flowchart illustrating the selection and research process for the research subjects of this invention. Figure 2 This invention presents the SERPING1 expression profile and the relationship between SERPING1 promoter methylation and mRNA expression. A shows SERPING1 expression in healthy controls (HCs), untreated chronic hepatitis B (CHB), and the NUC-treated-HBV group in GSE159413; B shows SERPING1 expression in chronic hepatitis B (CHB) and HBeAg serological conversion (SCHBV) groups in GSE224283; C shows SERPING1 mRNA expression in PBMCs of HBeAg-positive, HBeAg-negative, and healthy controls in the clinical cohort; D shows SERPING1 mRNA expression in the clinical cohort of HBeAg-positive (e... + HBeAg negative (e)- PMR comparison between SERPING1 PMR and healthy controls (HCs); E shows a Spearman scatter plot of SERPING1 PMR versus mRNA expression; Figure 3 The present invention illustrates changes in plasma C1-INH levels; A shows the C1-INH levels in HBeAg-positive, HBeAg-negative, and healthy controls; B shows the C1-INH levels in patients during the immune tolerance phase, HBeAg-positive immune active phase, inactive phase, and HBeAg-negative immune active phase; C shows the correlation between C1-INH and HBeAg levels. Figure 4 This invention demonstrates the predictive effectiveness of SERPING1 promoter methylation levels. Figure 5 This invention compares SERPING1-related indicators between the HBeAg serological conversion group and the non-conversion group in the 96-week follow-up training and validation cohorts. A shows the difference in SERPING1 mRNA expression level; B shows the difference in plasma C1-INH level; C shows the difference in SERPING1 promoter PMR. Figure 6 This is a nomogram of HBeAg serological conversion prediction for the training cohort of this invention. Figure 7 The graphs show the performance evaluation of the training and validation queue models of this invention; A is the ROC curve; B is the threshold value of the serological conversion rate of HBeAg-positive patients as distinguished by the model score; C is the calibration curve; and D is the decision curve. Detailed Implementation

[0026] To clearly illustrate the technical features of this solution, the invention will be described in detail below through specific embodiments and in conjunction with the accompanying drawings.

[0027] 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 this application.

[0028] The term "HBeAg serological conversion" refers to the disappearance of HBeAg and the conversion of anti-HBe (hepatitis B e antibody HBeAb) to positive in HBeAg-positive chronic hepatitis B patients. It is an important clinical observation indicator and also the indicator determined at the 96-week evaluation time point in this invention.

[0029] The present invention will be described in detail below through specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments without specific conditions should preferably refer to the guidelines given in this application, or may be performed according to experimental manuals or conventional conditions in the art, or by referring to experimental methods known in the art. Unless otherwise specified, all methods are conventional methods in the art. In the following specific embodiments, temperature and time parameters are involved, allowing for acceptable deviations due to instrument testing accuracy or operational precision. The equipment and raw materials used are all commercially available or commonly used in the art.

[0030] Example 1: Screening and Validation of SERPING1 Gene Expression Using Public Transcriptome Data

[0031] The PBMC transcriptome datasets GSE224283 and GSE159413 were obtained from the Gene Expression Omnibus database. GSE224283 includes 3 patients with chronic hepatitis B and 3 patients who underwent HBeAg seroconversion, used to explore changes in host immune genes associated with HBeAg seroconversion. GSE159413 includes 12 treatment-naïve patients with chronic hepatitis B, 13 patients treated with nucleoside (nucleotide) analogs, and 19 healthy controls, used to evaluate the relationship between treatment status and the expression of host immune-related genes. Due to the small sample size of these public datasets, they are used for candidate biomarker screening and directional validation, not for model performance estimation.

[0032] See Figure 2 Analysis results (A and B) showed that, compared with healthy controls, SERPING1 mRNA expression was increased in untreated chronic hepatitis B patients; SERPING1 expression was decreased in the nucleoside (nucleotide) analogue treatment group compared with the untreated group; and SERPING1 mRNA expression showed a decreasing trend in patients with HBeAg seroconversion in GSE224283, although the difference was not statistically significant. These results suggest that SERPING1 may be related to the disease status and treatment response of chronic hepatitis B.

[0033] Example 2: Application of SERPING1 gene promoter DNA methylation level in predicting HBeAg seroconversion in HBeAg-positive chronic hepatitis B patients 96 weeks after nucleoside (acid) analogue therapy.

[0034] I. Study Subjects and Clinical Cohort

[0035] The research flowchart is shown below. Figure 1The cohort included 244 patients with chronic hepatitis B who met the 2018 AASLD guidelines for the diagnosis and treatment of chronic hepatitis B, and 39 healthy controls with normal liver function, negative HBV serological markers, and no history of malignant diseases. Chronic HBV infection was defined as HBsAg positivity for more than 6 months; the age of the participants was 18-80 years.

[0036] Exclusion criteria included liver disease caused by other viral infections, liver disease of other etiologies, malignant diseases, and pregnancy. Of the 244 patients, 159 were HBeAg positive and 85 were HBeAg negative. The 159 HBeAg-positive patients received entecavir or tenofovir disoproxil fumarate and completed 96 weeks of follow-up. Subsequently, these 159 HBeAg-positive patients were randomly assigned in a 7:3 ratio to a training group (n = 111) and a validation group (n = 48) to assess and validate potential factors influencing HBeAg serological conversion. HBeAg serological conversion was defined as the disappearance of HBeAg and the appearance of anti-HBe.

[0037] Age, sex, and serum biochemical and virological parameters were collected from all subjects.

[0038] II. Peripheral blood sample processing

[0039] Three mL of peripheral blood was collected from each subject into an EDTA anticoagulant tube. Plasma was collected and stored after centrifugation; peripheral blood mononuclear cells were separated using Ficoll-Paque density gradient centrifugation. Total RNA and genomic DNA were extracted from peripheral blood mononuclear cells according to the TRIzol reagent instructions. Plasma, RNA, and DNA samples were stored at -80°C.

[0040] 2.1 Extraction of peripheral blood mononuclear cells

[0041] All participants fasted for at least 8 hours, and 5 mL of fasting peripheral venous blood was collected the following morning using vacuum blood collection tubes containing EDTA anticoagulant.

[0042] 2.1.1 Reagent Preparation

[0043] PBS solution, lymphocyte separation solution.

[0044] 2.1.2 Consumables Preparation

[0045] Pasteur pipettes, 1.5mL EP tubes, 2mL EP tubes, 15mL centrifuge tubes, and 10mL syringes.

[0046] 2.1.3 Experimental Procedure

[0047] (1) After collecting the blood sample, slowly invert the EDTA blood collection tube several times to ensure that the blood sample and anticoagulant are thoroughly mixed.

[0048] (2) Place the blood collection tube in a centrifuge and centrifuge at 3000 rpm for 5 minutes. After centrifugation, the blood sample will separate into two layers. Carefully aspirate the upper layer of pale yellow clear plasma using a Pasteur pipette and transfer it to a 2 mL EP tube. Immediately store the tube in a -80°C ultra-low temperature freezer for subsequent experiments.

[0049] (3) After removing the upper plasma layer, add an equal volume of PBS solution to the blood cell precipitate in the blood collection tube, and gently and slowly mix with a Pasteur pipette to fully resuspend the blood cells.

[0050] (4) Take a 15mL centrifuge tube and add 3mL of lymphocyte separation medium. Then, slowly add the resuspended blood sample obtained in step (3) along the wall of the centrifuge tube so that the blood sample floats on top of the lymphocyte separation medium. Take care not to disturb the surface of the separation medium during the operation. After that, place the 15mL centrifuge tube in a centrifuge and centrifuge at 2000 rpm for 20 minutes.

[0051] (5) After centrifugation, the liquid is clearly divided into four layers, from top to bottom: plasma layer, peripheral blood mononuclear cell (PBMC) layer, cell separation fluid layer, and red blood cell and granulocyte layer. Carefully aspirate the PBMC layer using a Pasteur pipette and transfer it to a new 15ml centrifuge tube.

[0052] (6) Add PBS solution to 10 mL of the PBMCs cell suspension obtained in step (5), gently pipette to mix, and centrifuge at 2000 rpm for 10 minutes.

[0053] (7) Discard the supernatant, add 1 mL of PBS solution to the precipitate, and repeatedly pipette to resuspend the cells at the bottom of the precipitate. Transfer the cell resuspended solution to a 1.5 mL EP tube. Add 0.5 mL of PBS solution to a 15 mL centrifuge tube, mix well, and then transfer it to a 1.5 mL EP tube to reduce cell residue. Finally, centrifuge at 3000 rpm for 5 minutes.

[0054] (8) Finally, discard the supernatant and the resulting precipitate is PBMCs, which should be stored in an ultra-low temperature freezer at -80℃ for later use.

[0055] III. SERPING1 mRNA Detection

[0056] RNA was reverse transcribed into cDNA using a reverse transcription kit, with β-actin as an internal control. Real-time quantitative PCR was performed using SYBR Green. The 10 μL reaction mixture consisted of: 5 μL SYBR Green, 3 μL nuclease-free water, 0.5 μL upstream primer, 0.5 μL downstream primer, and 1 μL cDNA. 2 ^-ΔΔCt The method calculates the relative expression level of SERPING1.

[0057] The primer sequences used for detection are as follows: SERPING1 RT-qPCR upstream primer: 5'-GACATTTCACTGCTCTAAGGCTCA-3' (SEQ ID NO.7) SERPING1 RT-qPCR downstream primer: 5'-CTCCGTTATTCCTCAAAGTAACCC-3' (SEQ ID NO.8) β-actin RT-qPCR upstream primer: 5'-ATGGGTCAGAAGGATTCCTATGTG-3' (SEQ ID NO.9) β-actin RT-qPCR downstream primer: 5'-CTTCATGAGGTAGTCAGTCAGGTC-3' (SEQ ID NO.10).

[0058] IV. Detection of SERPING1 gene promoter methylation

[0059] 4.1 DNA Extraction

[0060] 4.1.1 Reagent Preparation

[0061] Ttizol, chloroform (pre-cooled at 4℃), anhydrous ethanol (pre-cooled at 4℃), 75% ethanol, 0.1M sodium citrate solution, 80mM sodium hydroxide solution.

[0062] 4.1.2 Consumables Preparation

[0063] 1.5mL EP tubes, 10μL pipette tips, 200μL pipette tips, 1000μL pipette tips.

[0064] 4.1.3 Experimental Procedure

[0065] (1) Remove peripheral blood mononuclear cell samples stored at -80℃ and place them in an ice box to thaw slowly. After the cells are completely thawed, add 1 mL of Trizol lysis buffer to the EP tube and use a pipette to repeatedly resuspend and lyse the cells. After processing, transfer the EP tube to a 4℃ freezer for overnight storage.

[0066] (2) On the second day, 300 μL of pre-cooled chloroform was added to the lysis product, the mixture was shaken vigorously for 30 seconds, and allowed to stand at room temperature for 3 minutes. Then, the sample was placed in a centrifuge pre-cooled to 4°C and centrifuged at 12,000 rpm for 15 minutes.

[0067] (3) After centrifugation, the mixture separates into three layers from top to bottom: an RNA layer (colorless and transparent aqueous phase), a DNA layer (white membrane), and a protein layer (pink bottom layer). Remove all the aqueous phase above the DNA layer, and then add 300 μL of anhydrous ethanol. Slowly invert the mixture to mix, and let it stand at room temperature for 3 minutes. Centrifuge at 14800 rpm for 10 minutes at 4°C. After centrifugation, carefully discard the supernatant; a transparent DNA precipitate will be visible adhering to the bottom or wall of the tube.

[0068] (4) Add 1 mL of 0.1 M sodium citrate solution to the tube to resuspend the DNA precipitate, and incubate with shaking at room temperature for 30 minutes. Then, place the reaction tube in a centrifuge at 4 °C and centrifuge at 14800 rpm for 5 minutes. Discard the supernatant after centrifugation. This step needs to be repeated three times.

[0069] (5) Add 1 mL of pre-chilled 75% ethanol to the EP tube containing the DNA precipitate, and gently pipette or shake to fully suspend the precipitate. Shake for 20 minutes at room temperature, then centrifuge at 14800 rpm for 5 minutes at 4°C, and carefully discard the supernatant. This ethanol washing step needs to be repeated twice.

[0070] (6) After discarding the supernatant again, add 1 mL of anhydrous ethanol to resuspend the DNA precipitate, shake at room temperature for 20 minutes, and then centrifuge at 14800 rpm for 5 minutes at 4°C.

[0071] (7) After centrifugation, discard the supernatant and place the EP tube opening in a room temperature environment to allow the residual ethanol in the tube to evaporate naturally until the DNA dries. This process usually takes 5 to 15 minutes.

[0072] (8) After the DNA precipitate has dried, add 50 μL of 8 mM sodium hydroxide solution and gently pipette to promote dissolution. If dissolution is incomplete, add another 50 μL of sodium hydroxide solution until the precipitate is completely dissolved. Transfer the dissolved DNA solution to a sterile centrifuge tube and store it at -80°C for long-term storage.

[0073] (9) After DNA extraction, its concentration and purity were determined using an ultra-micro spectrophotometer. The instrument was zeroed using 8 mM sodium hydroxide solution. The DNA concentration, absorbance values ​​at 260 nm and 280 nm, and the A260 / A280 ratio were recorded. Throughout the measurement process, the DNA sample was kept on ice to maintain stability. After the measurement was completed, the DNA sample was quickly returned to a -80°C freezer for storage, ready for subsequent experiments.

[0074] 4.2 DNA bisulfite modification

[0075] 4.2.1 Reagent Preparation

[0076] EZ DNA Methylation-Gold™ Kit (including CT Conversion Reagent powder, M-Dilution Buffer, M-Dissolving Buffer, M-Wash Buffer concentrate, Zymo-Spin™ IC adsorption column and matching collection tube), nuclease-free water, and anhydrous ethanol.

[0077] Preparation of CT Conversion Reagent working solution: First, briefly centrifuge the brown, light-protected tube containing CT Conversion Reagent powder to ensure all powder gathers at the bottom. Under light-protected conditions, open the tube cap and add 900 μL of nuclease-free water, 300 μL of M-Dilution Buffer, and 50 μL of M-Dissolving Buffer in sequence. After adding the sample, continuously shake at room temperature for 10 minutes to ensure complete dissolution of the powder.

[0078] The preparation steps for M-Wash Buffer working solution are as follows: According to the proportion specified in the instructions, accurately measure 96 mL of anhydrous ethanol and add it to the M-Wash Buffer concentrate. Then, shake or stir thoroughly to mix it evenly. The resulting M-Wash Buffer working solution can be used directly in subsequent experiments.

[0079] 4.2.2 Consumables Preparation

[0080] 1.5mL EP tube, 0.2mL PCR tube, 10μL pipette tip, 200μL pipette tip, 1000μL pipette tip.

[0081] 4.2.3 Experimental Procedure

[0082] (1) Calculate the DNA sampling volume: Calculate the required DNA solution volume according to the formula DNA volume (μL) = 1000 / DNA concentration (ng / μL). If the DNA volume is ≥20μL, then take 20μL; if it is less than 20μL, then the actual sampling volume is rounded up according to the calculated result (for example, if the calculated result is 1.1μL, take 2μL), and the final sampling volume is not less than 2μL.

[0083] (2) Add 130 μL of CT Conversion Reagent working solution and the DNA solution calculated in step (1) to the PCR tube in sequence. If the DNA sample volume is less than 20 μL, use nuclease-free water to make up to 20 μL to ensure that the total liquid volume in the reaction system is 150 μL.

[0084] (3) Use a vortex mixer to thoroughly mix the PCR tube, then briefly centrifuge in a microcentrifuge to collect all liquid at the bottom of the tube. Place the PCR tube in the PCR instrument and set the reaction program in Table 1: Table 1 Reaction Program Settings

[0085] (4) Assemble the Zymo-Spin™ IC adsorption column with the matching collection tube. Add 600 μL of M-Binding Buffer to the adsorption column, followed by all the reaction products obtained in step (3). After thoroughly mixing the mixture with the M-Binding Buffer, centrifuge at 13400 rpm for 30 seconds. After centrifugation, discard the waste liquid in the collection tube.

[0086] (5) Add 100 μL M-Wash Buffer to the adsorption column, then centrifuge at 13400 rpm for 30 seconds, and discard the waste liquid in the collection tube after centrifugation.

[0087] (6) Add 200 μL M-Desulphonation Buffer to the adsorption column, let it stand at room temperature for 20 minutes, then centrifuge at 13400 rpm for 30 seconds, and discard the waste liquid in the collection tube again.

[0088] (7) Add 200 μL M-Wash Buffer to the adsorption column, centrifuge at 13400 rpm for 30 seconds, and discard the waste liquid in the collection tube. This washing step needs to be repeated twice. After the two washings are completed, discard the collection tube.

[0089] (8) Assemble the adsorption column with a new 1.5 mL EP tube. Add 20 μL M-Elution Buffer to the center of the adsorption column matrix, let stand for 1 minute, and then centrifuge at 13400 rpm for 30 seconds. The liquid collected in the EP tube after centrifugation is the bisulfite-modified DNA product. Store the product at -20°C for later use.

[0090] 4.3 MethyLight (methylation-specific quantitative PCR)

[0091] 4.3.1 Primer and probe synthesis

[0092] This study obtained the promoter region sequence of the SERPING1 gene by searching the NCBI database. Based on the design principles of methylation-specific probes and primers, methylation detection probes and corresponding primers suitable for MethyLight technology were designed using the MethPrimer online platform and Oligo 7 software. All designed products were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the products underwent necessary quality control verification.

[0093] The primer sequences are as follows: SERPING1 methylation-specific upstream primer: 5'-TTTTAGTAGTTGGGATTATATGTGC-3' (SEQ ID NO. 1) SERPING1 methylation-specific downstream primer: 5'-ATATAAAACACCCAACTCAACA-3' (SEQ ID NO. 2) SERPING1 methylation-specific TaqMan probe: 5'-AAACTAACCGAACACGATAACTCAC-3' (SEQ ID NO. 3); the 5' end is labeled with a FAM fluorescent group, and the 3' end is labeled with a BHQ1 quencher group; ACTB methylation-specific upstream primer: 5'-TGGTGATGGAGGAGGTTTAGTAAGT-3' (SEQ ID NO.4) ACTB methylation-specific downstream primer: 5'-AACCAATAAAACCTACTCCTCCCTTAAA-3' (SEQ ID NO. 5) ACTB methylation-specific TaqMan probe: 5'-ACCACCACCCAACACACAATAACAAACACA-3' (SEQ ID NO.6); the 5' end is labeled with a FAM fluorescent group, and the 3' end is labeled with a BHQ1 quencher group.

[0094] The primers and probes were provided in lyophilized powder form. Before use, they should be centrifuged at 4000 rpm for 5 minutes to ensure the powder settles completely at the bottom of the tube. Then, strictly following the product instructions, the powder was prepared into a 100 μM stock solution using nuclease-free water, and further diluted to a working concentration of 10 μM for subsequent MethyLight experiments. It is particularly important to note that the probes are photosensitive due to the presence of fluorescent groups; therefore, strict light protection measures must be taken throughout the entire preparation, dilution, and storage process.

[0095] 4.3.2 Reaction System

[0096] Table 2 MethyLight Reaction System

[0097] 4.3.3 Program Setup

[0098] Table 3 Methylation Detection Procedure

[0099] 4.3.4 Result Processing

[0100] The methylation level of the SERPING1 promoter region is expressed as the Percentage of the Methylated Reference (PMR). The calculation formula is as follows: PMR = 100% × 2^- [(Ct experimental sample SERPING1 gene - Ct experimental sample ACTB gene) - (Ct positive control SERPING1 gene - Ct positive control ACTB gene)] See Figure 2 The results of tests D and E showed that HBeAg-positive patients had lower SERPING1 promoter methylation levels compared to HBeAg-negative patients (P=0.0003). Correlation analysis showed that SERPING1 promoter methylation levels were negatively correlated with mRNA expression in patients with chronic hepatitis B (r=-0.2322, P=0.0003), supporting an epigenetic association between the two.

[0101] V. Clinical staging and SERPING1 mRNA / plasma C1-INH detection

[0102] According to the 2018 American Association for the Study of Liver Diseases guidelines, patients with chronic hepatitis B were divided into four categories: 46 patients in the immune-tolerant phase, 33 patients in the HBeAg-positive immune active phase, 68 patients in the inactive chronic hepatitis B phase, and 10 patients in the HBeAg-negative immune active phase. Another 87 patients who could not be classified into the above categories were placed in the gray zone.

[0103] Plasma C1-INH was quantitatively detected using an enzyme-linked immunosorbent assay (ELISA). A standard curve was established according to the kit instructions (Shanghai Langdun Biotechnology Co., Ltd.), and the absorbance was read at 450 nm. The absolute concentration of C1-INH in the sample was calculated based on the standard curve.

[0104] See Figure 3 The results showed that plasma C1-INH levels in HBeAg-positive patients were higher than those in HBeAg-negative patients (P=0.0051) and healthy controls (P=0.0149). C1-INH levels in patients in the immune-tolerant phase (Immune-tolerant CHB) and the HBeAg-positive immune-active phase (HBeAg(+)Immune-active CHB) were higher than those in the inactive phase (P values ​​were 0.0148 and 0.0129, respectively); C1-INH was positively correlated with serum HBeAg levels (r=0.1659, P=0.0107).

[0105] VI. Predictive Value of HBeAg Serological Conversion

[0106] 159 HBeAg-positive chronic hepatitis B patients received nucleoside (nucleotide) analogue therapy and were followed up for 96 weeks. Clinical testing for HBeAg seroconversion (HBeAg negative, anti-HBe positive) was performed on each patient, and 43 patients (27.04%) were confirmed to have HBeAg serological conversion, while 116 patients did not. Initial grouping was stratified according to outcome to ensure that the proportion of serological conversion in the training and validation cohorts was equal or similar to reduce bias. In the training cohort (111 patients), 30 patients underwent serological conversion, and 81 did not; in the validation cohort (48 patients), 13 patients underwent serological conversion, and 35 did not.

[0107] Based on the baseline characteristics of 159 HBeAg-positive chronic hepatitis B patients before receiving nucleoside (nucleotide) analogue therapy, the baseline characteristics of the confirmed serological conversion group and the non-conversion group were statistically analyzed in Table 4 (left side). In the training group, there was a significant difference in the methylation rate (PMR) of the SERPING1 promoter between patients who underwent serological conversion and those who did not. See [link to table]. Figure 4 In the middle left figure, the ROC curve (AUC) of the training group is 0.849 (95% CI: 0.761-0.937), the maximum Youden index is 0.678, corresponding to an optimal SERPING1 methylation level cutoff value of 34.16%, a sensitivity of 90%, and a specificity of 77.78%. In other words, a SERPING1 methylation level <34.16% indicates a high probability of model conversion or prediction, while a SERPING1 methylation level ≥34.16% indicates a low probability of model conversion or prediction not converted.

[0108] Table 4. Baseline characteristics of HBeAg serological conversion and non-conversion groups in the training and validation cohorts.

[0109] To verify the accuracy of the above predictions, validation was conducted in another validation cohort (n = 48). The results showed that the area under the ROC curve (AUC) of the validation cohort was 0.791 (95% CI: 0.661–0.921), indicating that the indicator has good predictive power. Figure 4 The image is shown in the middle right corner.

[0110] In both the training and validation cohorts, the converted group showed higher baseline SERPING1 mRNA expression and plasma C1-INH levels compared to the non-converted group, consistent with the findings in the fifth study. The SERPING1 promoter methylation PMR was also lower. (See details...) Figure 5 These results suggest that SERPING1 could be a candidate predictor of HBeAg serological conversion outcome at 96 weeks.

[0111] VII. Establishment of Predictive Model

[0112] Based on the methylation level of the SERPING1 promoter in the "sixth" section, a predictive model can be constructed by combining baseline alanine aminotransferase (ALT) and baseline quantitative HBeAg indicators, which can further improve predictive performance and clinical application value.

[0113] Using the actual outcomes of the aforementioned training cohort as the dependent variable and baseline SERPING1 promoter DNA methylation level (PMR), ALT, HBeAg, and other indicators as independent variables, a Logistic prediction model was established.

[0114] The Logistic prediction model includes a sample detection unit, a data input unit, a data processing unit, and a result output unit. The sample detection unit is used to obtain the baseline SERPING1 promoter methylation PMR. The data input unit is used to receive the PMR, baseline quantitative HBeAg, and alanine aminotransferase (ALT). The data processing unit calculates the probability P of HBeAg serological conversion at 96 weeks after the model parameters are verified and locked by the original regression output. The result output unit outputs the probability value.

[0115] The model in the above data processing unit uses Logistic multivariate regression and is converted into a nomogram, as shown below. Figure 6The formula for calculating the probability is as follows: logit(P) = β0 + β1 PMR(SERPING1) + β2×ALT + β3×HBeAg, P=1 / [1+e^(-logit(P))], where P is the HBeAg serological conversion probability at 96 weeks, β0 to β3 are model parameters confirmed by the original regression output and locked before application, β0=2.450305819, β1=-0.097796161, β2=0.012203862, β3=-0.002170063, and e is the base of the natural logarithm ≈2.718281828. Based on the research cutoff value, P≥0.320 is defined as a high probability of model conversion or predicted conversion, and P<0.320 is defined as a low probability of model conversion or predicted no conversion.

[0116] The above model was further validated in a validation queue to assess its predictive value. See also Figure 7 The graph shows the performance evaluation of the training and validation queue models. Specifically, evaluations using calibration curves and decision curves demonstrate that the prediction model exhibits good predictive performance.

[0117] The present invention has been described in detail above. The specific embodiments described above should not be construed as limiting the scope of protection of the present invention. Any alternative modifications or variations made to the embodiments of the present invention by those skilled in the art will fall within the scope of protection of the present invention.

[0118] Any aspects of this invention not described in detail are well-known to those skilled in the art.

Claims

1. Application of reagents for detecting SERPING1 gene promoter DNA methylation levels in the preparation of products for predicting or assisting in predicting HBeAg serological seroconversion in HBeAg-positive chronic hepatitis B patients after treatment.

2. The application according to claim 1, characterized in that, The assessment point for HBeAg serological seroconversion is 96 weeks after HBeAg-positive chronic hepatitis B patients receive nucleoside (acid) analogue therapy.

3. The application according to claim 1, characterized in that, The reagents include primers, probes, or nucleic acid chips.

4. The application according to claim 3, characterized in that, The reagent includes a primer pair and a probe for detecting the DNA methylation level of the SERPING1 gene promoter. The upstream primer sequence of the primer pair is shown in SEQ ID NO.1, the downstream primer sequence of the primer pair is shown in SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO.

3.

5. The application according to claim 1, characterized in that, A SERPING1 gene promoter DNA methylation level of <34.16% in the sample to be tested indicates a high probability or predicted conversion, while a level of ≥34.16% indicates a low probability or predicted no conversion.

6. The application according to claim 1, characterized in that, The product includes a kit or a biochip; the kit includes primer pairs and probes for detecting the methylation level of SERPING1 gene promoter DNA, genomic DNA extraction reagents, bisulfite modification reagents, methylation detection internal control primer pairs and probes, a fully methylated positive control, an unmethylated or template-free negative control, and real-time fluorescent PCR reaction reagents.

7. A prediction system, characterized in that, The prediction system is used to predict or assist in predicting the HBeAg serological seroconversion outcome in HBeAg-positive chronic hepatitis B patients after receiving nucleoside (acid) analogue therapy at 96 weeks. The prediction system includes a sample detection unit, a data input unit, a data processing unit, and a result output unit. The sample detection unit is used to obtain the baseline SERPING1 promoter methylation PMR. The data input unit is used to receive the baseline SERPING1 promoter methylation PMR, baseline quantitative HBeAg, and alanine aminotransferase (ALT) results. The data processing unit calculates the probability of HBeAg serological seroconversion at 96 weeks after the model parameters are verified and locked by the original regression output. The result output unit outputs the probability value.

8. The prediction system according to claim 7, characterized in that, The data processing unit uses a Logistic multivariate regression model, which is then converted into a nomogram. The model formula for calculating the probability is as follows: logit(P) = β0 + β1 PMR(SERPING1) + β2×ALT + β3×HBeAg, P=1 / [1+e^(-logit(P))], where P is the HBeAg serological conversion probability at 96 weeks, and β0 to β3 are model parameters confirmed by the original regression output and locked before application: β0=2.450305819, β1=-0.097796161, β2=0.012203862, β3=-0.002170063.

9. The prediction system according to claim 8, characterized in that, When P ≥ 0.320, the probability is high or a conversion is predicted; when P < 0.320, the probability is low or no conversion is predicted.

10. The prediction system according to any one of claims 7-9, characterized in that, The prediction process involves the following steps: S1: Obtain baseline clinical information and a specified volume of peripheral blood before the subject begins prescribed nucleoside (acid) analogue therapy; S2: Isolate peripheral blood mononuclear cells (PBMCs) and extract genomic DNA; S3: Perform bisulfite conversion on DNA, and evaluate the success of the conversion and amplification using specified internal controls and references; S4: Methylight reaction was performed using SERPING1 methylation-specific primers and TaqMan probes, with full methylation reference and internal reference used to calculate the SERPING1 promoter PMR; S5: Input the qualified PMR and baseline HBeAg and ALT into the following preset model formula: logit(P) = 2.450305819 - 0.097796161 × PMR + 0.012203862 × ALT -0.002170063 × HBeAg, P=1 / [1+e^(-logit(P))]; S6: Calculate and output the predicted probability of HBeAg serological conversion at week 96.

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