Application of biomarker in preparation of product for predicting nephrotic syndrome relief of MCD patient after glucocorticoid treatment

Abstract

The invention provides application of a biomarker in preparation of a product for predicting nephrotic syndrome remission of MCD patients after glucocorticoid treatment. The marker is selected from at least one of specific methylation sites, and the specific methylation sites comprise cg12655112, cg14642437, cg00483891 and cg00361017, and the specific methylation sites comprise a specific methylation site, a specific methylation site, a specific methylation site, a specific methylation site, a specific methylation site, a specific methylation site and a specific methylation site. By detecting the methylation level of the specific methylation site, accurate prediction of the glucocorticoid treatment effect of patients with minimal lesions can be realized, and a scientific basis is provided for individualized treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an application of a biomarker in the preparation of a product for predicting the remission of nephrotic syndrome in MCD patients after glucocorticoid treatment. Background Art

[0002] Minimal change disease (MCD) is an immune-mediated glomerular disease and one of the most common diseases leading to nephrotic syndrome (NS). Glucocorticoids (also known as steroid hormones, the English name is glucocorticosteroid, abbreviated as GC / GCs) are often used to treat minimal change disease. 90% of patients are sensitive to glucocorticoid treatment, but the recurrence rate of the disease is as high as 60%. Although clinical symptoms can provide certain clues, it is not enough to predict the remission of minimal change disease based on clinical symptoms alone. It is usually necessary to combine multiple examination methods and detection indicators for comprehensive evaluation. The existing technology does not have an effective detection indicator to predict the remission of nephrotic syndrome induced by glucocorticoid treatment in patients with minimal change disease. Summary of the invention

[0003] The purpose of the present invention is to provide a marker for predicting the remission of nephrotic syndrome in MCD patients after glucocorticoid treatment, and by detecting the methylation level of the marker, accurate prediction of the effect of glucocorticoid treatment on patients with minimal lesions can be achieved, providing a scientific basis for individualized treatment.

[0004] In order to achieve the above objectives, the present invention provides the use of a biomarker in the preparation of a product for predicting the remission of nephrotic syndrome in MCD patients after glucocorticoid treatment, wherein the biomarker is selected from at least one of the specific methylation sites, and the specific methylation sites include cg12655112, cg14642437, cg00483891 and cg00361017.

[0005] In a specific embodiment, the product is a kit, a gene chip or a high-throughput sequencing platform.

[0006] In a specific embodiment, the test sample of the product is DNA of a subject.

[0007] In a specific embodiment, the product includes a detection reagent for detecting the methylation level of at least one specific methylation site.

[0008] In a specific embodiment, the detection reagent also includes a specific probe corresponding to the specific methylation site to be detected and a PCR reaction solution.

[0009] In a specific embodiment, demethylation of cg12655112, cg14642437, cg00483891 and cg00361017 among the specific methylation sites predicts a high probability of the subject using glucocorticoid-induced remission of nephrotic syndrome in patients with minimal change disease.

[0010] In a specific embodiment, cg12655112, cg14642437, cg00483891 and cg00361017 among the specific methylation sites are positive regulation sites.

[0011] In a specific embodiment, when the methylation level of the specific methylation site is low compared to the baseline, it is predicted that the subject has a high probability of using glucocorticoids to induce remission of nephrotic syndrome in patients with minimal change disease.

[0012] The beneficial effects of the present invention include at least:

[0013] 1. The present invention discovers for the first time that after long-term glucocorticoid treatment in MCD patients, the patient's specific methylation sites cg12655112, cg14642437, cg00483891 and cg00361017 are differentially expressed. In this way, the specific methylation sites can be used as markers to predict the remission of nephrotic syndrome in MCD patients after glucocorticoid treatment, and to assist in evaluating whether glucocorticoid treatment is effective for MCD patients. This provides a new approach to predicting the prognosis of MCD patients treated with glucocorticoids, thereby providing a scientific basis for individualized treatment.

[0014] 2. The present invention detects the methylation level of specific methylation sites through detection reagents, and can evaluate the relief of nephrotic syndrome in patients with minimal change disease after treatment with glucocorticoids, so as to guide the clinical application of glucocorticoids.

[0015] 3. The detection reagents for detecting the methylation level of specific methylation sites are applied to the test kit, which can quickly predict whether the glucocorticoid treatment is effective through the relief of nephrotic syndrome, and monitor and evaluate the response of the immune system at any time, so as to promote the outcome of the disease by adjusting the treatment plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 KEGG analysis of genes that may be regulated by methylation at the 8th month;

[0017] Figure 2 This is the volcano plot of methylation regulation changes caused by conventional GC treatment for 2 months;

[0018] Figure 3 This is the volcano plot of methylation regulation changes caused by conventional GC treatment for 4 months;

[0019] Figure 4 This is the volcano plot of methylation regulation changes caused by 8 months of conventional GC treatment;

[0020] Figure 5 This is a graph showing changes in methylation levels of specific methylation sites caused by conventional GC treatment for 2, 4, and 8 months;

[0021] Figure 6 This is a graph showing the changes in methylation levels of specific methylation sites after 2, 4, and 8 months of conventional GC treatment compared with the baseline (control);

[0022] Figure 7 ROC curves for sensitivity and specificity of specific methylation sites;

[0023] Figure 8 This is one of the maps showing changes in methylation regions at specific methylation sites during glucocorticoid treatment;

[0024] Fig. 9 This is the second diagram of the changes in methylation regions at specific methylation sites during glucocorticoid treatment. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0026] The experimental methods without specific conditions in the examples are usually carried out under conventional conditions or conditions recommended by the manufacturers. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0027] Related technologies

[0028] The glucocorticoid (GC) signaling pathway is the molecular mechanism of cellular response to GC. Most GCs bind to corticosteroid binding globulin (CBG) in plasma, enter the cell through passive diffusion, and bind to the glucocorticoid receptor (GR) in the cell. GR is a nuclear receptor. After binding to GC, its conformation changes, exposing the nuclear localization signal. The GR-GC complex then transfers to the nucleus and binds to the glucocorticoid response element (GRE) on the promoter of the target gene in the form of homodimers or heterodimers, recruiting a series of auxiliary factors that regulate chromatin remodeling, activating or inhibiting specific gene transcription. Previous studies have shown that glucocorticoids participate in epigenetic mechanisms, which are usually mediated by histone modification, DNA methylation, and microRNA. Glucocorticoids not only regulate the expression of miRNAs through glucocorticoid receptors in a variety of physiological and pathological processes, but also can act together by changing the expression of DNA methyltransferases (DNMTs), inducing DNA demethylation, affecting histone modifications, and changing the expression of glucocorticoid receptors, so that glucocorticoids can induce or inhibit specific gene expression in different cell types and tissues. After searching, it was found that methylation sites have long been used as biomarkers for the diagnosis and prognosis monitoring of various kidney diseases such as nephrotic syndrome, diabetic nephropathy, IgA nephropathy, and membranous nephropathy. However, there are no related studies on methylation sites for biomarkers that are significantly correlated with the remission of nephrotic syndrome in patients with minimal change disease disclosed in existing domestic and foreign technologies, nor have studies on biomarkers for predicting the remission of nephrotic syndrome induced by glucocorticoids. Based on the important role of genomic and epigenetic changes in the progression of minimal change disease, and the fact that glucocorticoids may reverse these changes after the treatment of minimal change disease, the present invention tracked the methylation of specific sites in 8 patients with minimal change disease at 2, 4, and 8 months after the treatment of nephrotic syndrome induced by glucocorticoids, and thus launched a detection kit for predicting the treatment effect of minimal change disease. This invention is of great significance for predicting the disease outcome of patients with minimal change disease after glucocorticoid treatment.

[0029] The present invention provides an application of a biomarker in the preparation of a product for predicting the remission of nephrotic syndrome in MCD patients after glucocorticoid treatment, wherein the marker is selected from at least one of specific methylation sites, and the specific methylation sites include cg12655112, cg14642437, cg00483891 and cg00361017.

[0030] The coordinate information of specific methylation gene sites is shown in Table 1.

[0031] Table 1 Coordinate information of methylation sites

[0032] Methylation site Gene name Chromatin ID area Location cg12655112 EHD4 chr15 Body-shelf 41968956-41968957 cg14642437 FKBP5 chr6 5'UTR-shelf 35684744-35684745 cg00483891 CCDC115 chr2 Body-shore 130340434-130340435 cg00361017 PDE10A chr6 Body-shore 165660178-165660179

[0033] It should be noted that the coordinate information of specific methylation sites is based on the sequence of the human reference genome Hg19.

[0034] Preferably, the test sample of the product is DNA of a subject.

[0035] Preferably, the product is a kit, a gene chip or a high-throughput sequencing platform.

[0036] When the product is a kit, in addition to the detection reagent, the kit may also include other conventional reagents required for methylation quantitative PCR, such as one or more of DNA extraction reagents, sulfite, deionized water, Taq mixture buffer, etc. Since such commonly used reagents for methylation quantitative PCR can be purchased separately from the market or prepared by oneself, which reagents need to be assembled into the kit can be configured according to the actual needs of the customer, and for convenience, all of them can be assembled into the kit.

[0037] Preferably, the product comprises a detection reagent for detecting the methylation level of at least one specific methylation site.

[0038] Preferably, the detection reagent also includes a specific probe corresponding to the specific methylation site to be detected and a PCR reaction solution.

[0039] In the present invention, the design of specific probes and PCR primers in the PCR reaction solution is a conventional technique in the art, and other sequences may also be designed. For example: the design of probes for specific methylation sites is shown in Table 2.

[0040] It should be noted that those skilled in the art can design primer pairs for amplification based on the coordinate information of specific methylation sites and the coordinates of the disclosed target sites. The design of primer pairs belongs to the prior art and will not be described in detail here.

[0041] Table 2 Probe sequences for specific methylation sites

[0042]

[0043] Preferably, cg12655112, cg14642437, cg00483891 and cg00361017 among the specific methylation sites are positive regulation sites.

[0044] Preferably, when the methylation level of the specific methylation site is low compared to the baseline, it is predicted that the subject has a high probability of using glucocorticoids to induce remission of nephrotic syndrome in patients with minimal change disease.

[0045] It should be noted that, in the present invention, the baseline is a standard for judging whether the expression level is high expression or low expression, and can be understood as a standard expression level.

[0046] Example 1

[0047] Screening and validation of DNA methylation markers for predicting remission of nephrotic syndrome in patients with minimally invasive disease induced by glucocorticoid therapy

[0048] 1.1. Prospective time series studies

[0049] Eight patients diagnosed with minimal change disease (MCD) and with a first episode of nephrotic syndrome (NS) were selected for follow-up for 8 months. In this time series study, all patients were fully informed and received a standard conventional glucocorticoid treatment regimen, which started with high-dose oral prednisone (1 mg / kg / day, up to 80 mg / day) or equivalent doses of methylprednisolone (0.8 mg / kg / day) for 6 to 8 weeks, and reduced by 5 mg every two weeks after complete remission. The included patients did not receive any immunosuppressive therapy other than glucocorticoids. Patients who failed to achieve complete remission of nephrotic proteinuria within 8 weeks or had early relapse were excluded from this study and received other immunosuppressive therapy.

[0050] 1.2 Research Methods

[0051] Methylation sequencing was performed according to the protocol of Shanghai Biochip Co., Ltd. The Methylation EPIC Magnetic Bead Chip uses Illumina Magnetic Bead SequenceTM Infinite Technology to specifically identify methylation sites. In the Methylation EPIC Magnetic Bead Chip, both InfiniumⅠ and InfiniumⅡ probe designs are used to maximize the detection range.

[0052] For each methylation site, two probes are designed: M-type magnetic beads and U-type magnetic beads. The tail of the M-type magnetic beads is G, which is used to detect methylation sites. The tail of the U-type magnetic beads is A, which is used to detect unmethylated sites. If a certain site on the genome is methylated, then under the treatment of bisulfite, GC remains GC, and paired with M-type magnetic beads, a fluorescent signal can be detected after the incorporation of fluorescently labeled nucleotides. The M-type magnetic beads emit light. On the contrary, if it is not methylated, then under the treatment of bisulfite, GC becomes GT, paired with U-type magnetic beads, and the U-type magnetic beads emit light after extension.

[0053] 1.3 Experimental procedures

[0054] 1. DNA extraction includes the following steps: 1. Add 20μ proteinase K to a 1.5ml centrifuge tube, add 5-10μl anticoagulant blood, and adjust the volume to 220μl with PBS; 2. Add 200μl anhydrous ethanol and shake to mix; 3. Add all the mixed solution (including the precipitate) in step 2.3 to the mini spin column of the DNA kit, centrifuge at ≥6000xg (8000rpm) for 1 minute, and discard the flow-through liquid and collection tube; 4. Place the column in a new centrifuge collection tube, add 500μl buffer AW1, ≥6000xg (800 0rpm) for 1 minute, discard the flow-through liquid and the collection tube; 5. Place the column in a new centrifuge collection tube, add 500μl buffer AW2, centrifuge at 16100xg (13200rpm) for 3 minutes, discard the flow-through liquid and the collection tube; 6. Place the column in a new 1.5ml centrifuge tube, add 200μl buffer AE to the membrane in the column, place at room temperature for 1 minute, and centrifuge at ≥6000xg (8000rpm) for 1 minute to elute; 7. Repeat step 7 to obtain the maximum DNA yield; 8. DNA quantification and quality inspection.

[0055] 2. The treatment of gDNA (gene DNA) with bisulfite includes: 1. Affix a BCD barcode label to each new 96-well microplate without a skirt (0.2 ml) for marking; 2. Denature the gDNA according to the requirements of the EZDNA Methylation-Gold Kit, and then add the conversion reagent; 3. Incubate in a thermal cycler under the following conditions: 98°C for 10 minutes; 64°C for 2.5 hours; 4°C permanently (up to 20 hours); 4. After completion, the temperature of the thermal cycler is maintained at 4°C, and the gDNA is placed in it for use.

[0056] 3. Preparation of BCD includes: 1. According to the requirements of the kit, perform the following steps in sequence: a. Wash to remove the conversion reagent, b. Incubate in desulfonation buffer at room temperature for 15 minutes, c. Wash to remove the desulfonation buffer, d. Add elution buffer; 2. Centrifuge to elute gDNA; 3. Transfer the gDNA treated with bisulfite into the BCD plate; 4. Freeze the sealed BCD for later use at -80°C.

[0057] IV. Alkaline denaturation-full genome amplification includes: 1. Paste the MSA4 barcode label on the new MIDI plate for marking; 2. Add 20μl MA1 to the MSA4 plate well; 3. Add 4μl of DNA sample treated with bisulfite to the MSA4 plate well, and record the corresponding sample ID in the MSA4 plate well on the laboratory tracking form; 4. Add 4μl 0.1mol / L NaOH to the MSA4 plate well, and cover the 96-well plate cover (pay attention to the correct direction of the letters when covering, and cover tightly to prevent splashing, volatilization and contamination) ; 5. Mix on an oscillator at 1600rpm for 1 minute; centrifuge at 280xg for 1 minute; 6. Incubate at room temperature and let stand for 10 minutes; 7. Add 68μlRPM to the MSA4 plate wells; 8. Add 75μlMSM to the MSA4 plate wells; 9. Replace the cover pad; 10. Turn the covered MSA4 upside down at least 10 times to mix thoroughly; 11. Centrifuge at 280xg for 1 minute; 12. Incubate in a hybridization oven at 37°C for 22 hours overnight (20 to 24 hours).

[0058] 5. The steps of breaking-precipitation-resuspending-hybridization include: 1. Take out MSA4 and centrifuge at 50xg for 1 minute; 2. Add 50μl FMS to the MSA4 plate well and cover it again; 3. Mix on an oscillator at 1600rpm for 1 minute; centrifuge at 50xg for 1 minute; 4. Incubate in a 37℃ heat resistor for 1 hour; 5. The following steps are selected: (1) Continue the experiment; (2) Seal the MSA4 and freeze it for later use, and precipitate it at -15℃ to -25℃; 6. Open the cover of MSA4, add 100μl PM1 to the MSA4 plate well, and cover it again; 7. Mix on an oscillator at 1600rpm for 1 minute; 8. 10. Add 300μl 100% isopropanol to the MSA4 plate wells and replace with new dry cover pads; 11. Turn the covered MSA4 upside down at least 10 times to mix thoroughly; 12. Incubate in a 4℃ refrigerator for 30 minutes; 13. Centrifuge at 3000xg in a centrifuge at 4℃ for 20 minutes; then immediately take the MSA4 out of the centrifuge, discard the cover pad, immediately turn it over to remove the supernatant, turn it upside down horizontally on the absorbent paper and tap it until there is no watermark, which takes about 1 minute. Do not move the blue precipitate and keep the MSA4 upside down.(If there is a delay after the centrifugation and the supernatant cannot be removed in time, it is necessary to centrifuge again for 20 minutes); 14. Keep the MSA4 in an upside-down state on the test tube rack and air-dry for 1 hour; 15. Choose from the following steps: (1) Continue the experiment; (2) Seal the MSA4 and freeze it for later use, and resuspend it at -15℃~-25℃; 16. Add 46μl RA1 to the MSA4 plate well, and freeze it after RA1 is used; 17. After covering the MSA4 with metal foil, heat the MSA4 down for 5 seconds, then change the MSA4 up and down order and heat it for another 5 seconds. Then lift the metal foil slightly to verify the sealing effect; 18. Place the sealed MSA4 in a 48℃ hybridization oven and incubate for 1 hour; 19. Mix on an oscillator at 1800rpm for 1 minute; centrifuge at 280xg for 1 minute. If the MSA4 is frozen, it needs to be repeatedly shaken and centrifuged until the blue precipitate is completely resuspended; 20. Choose from the following steps: (1) Continue the experiment; (2) Freeze the sealed MSA4 for later use at -15℃ to -25℃. If it is to be used for more than 24 hours, freeze it at -80℃; 21. Open the hybridization box and hybridize 1. Place the hybridization box liner in the hybridization box, add 400μl PB2 into the upper and lower small grooves of the hybridization box (the upper and lower small grooves correspond to one chip), close the hybridization box immediately to prevent evaporation, buckle it in the diagonal direction, and place it at room temperature for use; 22. After resuspending and sealing the MSA4, put it in a 95℃ heat resistor for denaturation for 20 minutes; 23. Take out the chip and place it at room temperature without opening it; 24. Cool the denatured MSA4 at room temperature for 30 minutes, and then centrifuge it at 280xg for 1 minute in a centrifuge; 25. Take the chip out of the box and carefully take it out of the package without touching the chip 26. Add 15μl DNA sample to the chip sample area according to the chip sample area sequence. Be careful not to generate bubbles during the slow flow, and record the corresponding number of the sample and chip. 27. Place the Hyb chamber slot with the chip in the hybridization box at the corresponding barcode position, and close the hybridization box. 28. Incubate in the hybridization oven at 48℃ for 18 hours overnight (16 to 24 hours). 29. Add 330ml 100% ethanol to XC4, mix vigorously for 15 seconds, and leave at room temperature overnight for use.

[0059] 6. Washing-extension-staining-scanning steps include: 1. Place the washing rack in the first wash chamber and completely submerge it in PB1; 2. Take out the Hyb chamber slot with the chip in the hybridization box, take out and insert the chip, remove the IntelliHyb seal and immediately place the chip in the washing rack; 3. Pull the washing rack with the chip up and down for 1 minute, be careful to be slow and gentle, and leave the liquid surface each time; 4. Place the washing rack with the chip in the second wash chamber, and pull it up and down for 1 minute as in the previous step; 5. Assemble the flow chamber: place the black frame in the microbead chip calibration and positioning frame, place the chip in the black frame (note that the chip is completely submerged in PB1), then correctly place the transparent plastic gasket, then place the calibration rod above the fixing frame, then correctly place the clean glass back electrode (the bevel corresponds to the chip barcode, and there is no bubble between the chip), and then correctly place the clamp at both ends, with a difference of 5mm from the upper and lower edges; 6. Take out the flow chamber, cut off the excess gasket, and place the flow chamber horizontally until the next step. 7. When the Te-Flow temperature is stabilized at 44°C, place the flow chamber correctly on the chamber rack with the chip barcode on the top. 8. Add the following sequence to the inclined surface of the glass back electrode: (1) 150 μl RA1, incubate for 30 seconds, repeat 5 times; (2) 450 μl XC1, incubate for 10 minutes; (3) 450 μl XC2, incubate for 10 minutes; (4) 200 μl TEM, incubate for 15 minutes; (5) 450 μl 95% formamide / 1 mM EDTA, incubate for 1 minute, repeat once; (6) incubate for another 5 minutes; (7) Adjust the Te-Flow temperature to 32°C (consistent with the temperature required by STM); (8) 450 μl XC3, incubate for 1 minute, repeat once; (9) Wait for the Te-Flow temperature to stabilize at 32°C. 9. Open iScan. 10. Add the following sequence to the inclined surface of the glass back electrode: (1) 250 μl SEM, incubate for 10 minutes; (2) 450 μl XC3, incubate for 1 minute, repeat once, and then incubate for 5 minutes; (3) 250 μl ATM, incubate for 10 minutes; (4) 450 μl XC3, incubate for 1 minute, repeat once, and then incubate for 5 minutes;

[0060] (5) 250μl SEM, incubate for 10 minutes; (6) 450μl XC3, incubate for 1 minute, repeat once, and incubate for 5 minutes; (7) 250μl ATM, incubate for 10 minutes; (8) 450μl XC3, incubate for 1 minute, repeat once, and incubate for 5 minutes; (9) 250μl SEM, incubate for 10 minutes; (10) 450μl XC3, incubate for 1 minute, repeat once, and incubate for 5 minutes. 11. Take out the flow chamber from the chamber rack and place it horizontally. 12. Add 310ml PB1 to the first butterfly wash and place the staining rack in it. 13. Remove the flow chamber, take out the chip, and place it in the staining rack. 14. Lift the staining rack with the chip up and down 10 times, be careful to be slow and gentle, leave the liquid surface each time, and then soak for 5 minutes. 15. After vigorous shaking of XC4, add 310 ml of XC4 to the second dishwash, take the staining rack out of the first dishwash and place it in the second dishwash. 16. Lift the staining rack with the chip up and down 10 times, be careful to be very slow and gentle, and leave the liquid surface each time, and then soak for 5 minutes. 17. After taking out the staining rack with the chip, immediately place the chip horizontally on the test tube rack with the front side facing up. 18. Take the chip out of the staining rack and place it horizontally on the test tube rack with the chip barcode facing up. 19. Dry the chip in a 508 mmHg (0.68 bar) vacuum pump for 50 to 55 minutes. 20. Wipe the dried XC4 on the back and both sides of the chip with a wipe paper to ensure that the back of the chip is flat. 21. Choose from the following steps: (1) Continue the experiment and scan with iScan; (2) Place the chip in its original packaging, place it in a vacuum pump at room temperature, and scan with iScan within 72 hours. 22. Start the iScan software to scan the chip: (1) Download the chip decoding file (.dmap) in advance; (2) Download the chip manifest file (.bpm) in advance; (3) The scanning takes about 1 hour.

[0061] 1.4 Statistical analysis

[0062] The raw data of the chip were first preprocessed using the R software minutefi package, and then the R software IMA package was used to screen the differences in methylation sites and methylation regions between sample groups. The entire methylation analysis process is divided into four steps: 1. Data quality control 2. Preprocessing 3. Methylation difference site analysis 4. Differential methylation region analysis The Beta value (β) is generally used as an indicator to measure the methylation degree of the site. The value range is (0,1). The closer it is to 1, the higher the methylation degree of the site, and the closer it is to 0, the lower the methylation degree of the site. The formula for Beta is as follows:

[0063]

[0064] 1.5. KEGG network construction

[0065] The PPI network was constructed using the STRING database, and key proteins were screened based on metrics. The Cytoscape software was used for visualization, and the KEGG network analysis was performed using the ClueGO plug-in in Cytoscape.

[0066] Figure 1 The KEGG analysis of genes that may be regulated by methylation at the 8th month describes the interaction between different proteins in the cell and the pathological and physiological processes involved. For example, Figure 1 This indicates that CD247 is involved in both the differentiation of Th1 and Th2 cells and the cytotoxicity mediated by natural killer cells; and that the differentiation of Th1 and Th2 cells is regulated by both CD247 and IL2RB.

[0067] 1.6 Experimental Results

[0068] The present invention conducted an eight-month methylation sequencing analysis on patients receiving glucocorticoid treatment, and used a volcano plot to display the number of genes regulated by methylation at different time points. In addition, the volcano plot also shows the changes in methylation at specific sites during 2, 4, and 8 months of hormone treatment. In the eighth month, more significant gene demethylation levels were observed, highlighting the long-term effects of methylation during glucocorticoid treatment. For details on the volcano plot, please refer to Figure 2 to Figure 4 , Figure 2 This is a volcano diagram of methylation regulation changes caused by conventional GC treatment for 2 months. Figure 3 This is a volcano diagram of methylation regulation changes caused by conventional GC treatment for 4 months. Figure 4 This is a volcano diagram of the changes in methylation regulation caused by conventional GC treatment for 8 months. Figure 2 to Figure 4 It can be seen that at least two significant changes were detected at the specific methylation site at the three detection time points.

[0069] Figure 5 This is the change in methylation level caused by conventional GC treatment for 2, 4, and 8 months. Figure 6 The changes in the methylation levels of the above methylation sites after 2, 4, and 8 months of conventional GC treatment compared with the baseline (control). Among the above 4 methylation sites, the methylation levels of cg12655112, cg14642437, and cg00483891 methylation sites changed significantly in 2 and 4 months, and the methylation levels of cg00361017 methylation sites changed significantly in 4 and 8 months.

[0070] Figure 7The ROC curve of the selected specific methylation sites was plotted. The AVG_Beta values ​​of the corresponding methylation sites in the original methylation sequencing data of 3 typical patients at 12 detection time points were selected and divided into two groups: disease state and remission state. The disease state corresponded to the AVG_Beta values ​​before treatment in 3 groups, and the remission state corresponded to the AVG_Beta values ​​after 2, 4, and 8 months of glucocorticoid treatment in 9 groups. The areas under the curve corresponding to the 4 methylation sites were cg12655112-0.889, cg14642437-0.926, cg00483891-0.852, and cg00361017-0.889, respectively, indicating that these monitoring points have high sensitivity and specificity.

[0071] A time series analysis of genome-wide DNA methylation was performed on individuals 2 months, 4 months, and 8 months before and after GC treatment. Figure 8 and Fig. 9 The regions that undergo methylation or demethylation during glucocorticoid treatment. Figure 8 and Fig. 9 It can be seen that the methylation changes are mainly concentrated in the body and opensea regions. The body refers to the coding region of the gene (i.e., exon) or the gene body, relative to the promoter region; opensea refers to those regions in the genome that are less tightly bound to proteins and may be more easily accessed by enzymes and methylated.

[0072] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions and substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention.

Claims

1. The use of a biomarker in the preparation of a product for predicting the remission of nephrotic syndrome in MCD patients after glucocorticoid treatment, characterized in that: The biomarker is selected from at least one of the specific methylation sites, and the specific methylation sites include cg12655112, cg14642437, cg00483891 and cg00361017.

2. The use according to claim 1, characterized in that: The product is a kit, a gene chip or a high-throughput sequencing platform.

3. The use according to claim 1, characterized in that: The test sample of the product is the DNA of the subject.

4. The use according to claim 1, characterized in that: The product includes a detection reagent for detecting the methylation level of at least one specific methylation site.

5. The use according to claim 4, characterized in that: The detection reagent also includes a specific probe corresponding to the specific methylation site to be detected and a PCR reaction solution.

6. The use according to claim 1, wherein demethylation occurs at cg12655112, cg14642437, cg00483891 and cg00361017 in the specific methylation sites, and the probability of the subject using glucocorticoid-induced minimal change nephrotic syndrome remission is high.

7. The use according to claim 6, characterized in that: Among the specific methylation sites, cg12655112, cg14642437, cg00483891 and cg00361017 are positive regulation sites.

8. The use according to claim 7, characterized in that: When the methylation level of the specific methylation site is low expressed relative to the baseline, it is predicted that the subject has a high probability of using glucocorticoid to induce remission of nephrotic syndrome in patients with minimal change disease.

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