Use of 5-hydroxymethylcytosine

Abstract

The present invention discloses a use of 5-hydroxymethylcytosine, and provides an application of 5-hydroxymethylcytosine in the diagnosis of systemic lupus erythematosus or in the preparation of a product for the diagnosis of systemic lupus erythematosus; the hydroxymethylation biomarker is one or more loci of ETS1. The present invention has the characteristics of strong sensitivity and high specificity, and can be applied to the auxiliary diagnosis of clinical systemic lupus erythematosus patients. By combining other clinical symptoms, signs and laboratory indicators, the diagnosis rate of early systemic lupus erythematosus can be improved, and clinicians can be assisted in formulating treatment plans in a timely manner, improving the diagnosis and treatment level of SLE and improving patient prognosis. In addition, the present invention also discloses a kit for diagnosing the above-mentioned biomarkers of SLE patients, which is intended to comprehensively improve the ability of clinicians to perform early diagnosis and preemptive intervention treatment on SLE patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular markers for testing molecular materials by means of chemical or physical properties of the test materials, and particularly relates to a use of 5-hydroxymethylcytosine. Background Art

[0002] Systemic lupus erythematosus (SLE) is a complex and heterogeneous systemic autoimmune disease characterized by chronic inflammation, immune complex deposition, and multisystem damage in affected organs. As a disease with complex and diverse clinical manifestations, the etiology of SLE remains unclear. Currently, it is generally believed to be the result of a combination of factors, including genetic susceptibility, environmental factors, hormonal changes, and socioeconomic factors. SLE is a major social and public health issue. Drug therapy and multidisciplinary comprehensive treatment can only control symptoms and delay disease progression, but cannot completely cure it. Improving the ability to diagnose SLE early to enable effective treatment is crucial. Therefore, biomarkers, particularly hydroxymethylation biomarkers, have emerged to help better diagnose SLE and assess its pathophysiology, with the ultimate goal of controlling disease progression and improving patient prognosis.

[0003] The current diagnosis of SLE is primarily based on complex criteria established by the American College of Rheumatology (ACR), including clinical manifestations, laboratory tests, histopathology, and imaging. However, laboratory test indicators for diagnosing SLE remain limited. Due to the wide heterogeneity of clinical manifestations and the lack of unified diagnostic criteria, SLE patients are often missed or misdiagnosed, resulting in a significant increase in patient mortality. Therefore, the search for new and reliable biomarkers and the construction of biomarker-based diagnostic kits are of great significance for the diagnosis of SLE. Previous studies have shown that epigenetics provides important information on the heterogeneity and pathophysiology of SLE. DNA methylation is one of the most studied epigenetic markers and plays a key role in various autoimmune diseases by affecting gene expression. 5-Hydroxymethylcytosine (5hmC), as an important product of the DNA demethylation process, provides new insights into the diagnosis of SLE.

[0004] 5hmC, as a stable epigenetic modification, is a key intermediate in active and passive DNA demethylation mediated by Ten-eleven translocation (TET) proteins. 5hmC modification plays an important role in cell development, differentiation, maturation and self-renewal. TET proteins, including TET1, TET2 and TET3, are a newly discovered family of DNA demethylases that can convert 5mC into 5hmC, which then catalyzes the generation of cytosine, ultimately leading to DNA demethylation and gene activation. 5hmC labeling can effectively screen patients with stable coronary heart disease at an early stage, and can distinguish normal healthy people from patients with stable coronary heart disease with high sensitivity and specificity, thereby effectively diagnosing patients with acute myocardial infarction. In another study, researchers evaluated the level of 5hmC by immunofluorescence and found that the level of 5hmC modification was increased in epithelial cells of patients with Sjögren's syndrome, while the level of 5hmC was decreased in inflammatory cells. In addition, the total 5hmC content in a variety of human cancers was significantly reduced compared with normal tissues adjacent to the cancer. In addition, some studies have found that the CD4 + Significant abnormal 5hmC modification is observed in promoter regions across the entire genome in T cells, suggesting a potential association between abnormal 5hmC modification and remission and relapse in SLE. 5hmC modification may play a crucial role in the pathogenesis of autoimmune diseases, including SLE, providing a new auxiliary diagnostic method. Therefore, identifying highly sensitive and specific hydroxymethylation biomarkers is a critical clinical challenge.

[0005] Cell-free DNA (cfDNA) in plasma originates from DNA released after damaged and ruptured cells, cleaved into fragments of approximately 160 nt by nucleases. As a complementary approach to imaging and tissue biopsy, "liquid biopsy" techniques based on cfDNA detection have garnered significant attention in various areas, including prenatal diagnosis, cancer screening, early diagnosis, treatment monitoring, and prognosis assessment. In recent years, cfDNA testing has been increasingly applied to disease diagnosis. cfDNA from patients with diabetic retinopathy (DR) contains 5hmC information that can be used to detect DR; 5hmC in cfDNA has recently been identified as a potential biomarker for non-small cell lung cancer and esophageal cancer. Circulating cfDNA biomarkers based on 5hmC are highly predictive for colorectal and gastric cancers, suggesting their potential as non-invasive diagnostic markers. Rheumatoid arthritis (RA) potentially releases cfDNA during inflammation, and plasma cfDNA can serve as a marker of disease progression in RA patients. These studies suggest that changes in 5hmC modification signals in cfDNA may also have potential value in the diagnosis and progression monitoring of SLE.

[0006] Current clinical diagnostic and classification criteria for SLE lack sensitivity for early diagnosis. Many patients present with atypical SLE symptoms early on, only to develop other overt manifestations as the disease progresses, leading to delayed treatment and a prolonged course of the disease. As the disease progresses, SLE can harm the kidneys and brain, posing a serious threat to patients' lives. Therefore, efforts are underway to develop early, objective, and simple diagnostic kits to facilitate early diagnosis and intervention of SLE. Hydroxymethylation biomarkers have shown potential in disease screening, diagnosis, personalized treatment, and prognostic assessment. The enrichment of 5hmC in plasma cfDNA across various genes in SLE patients may play a significant role in the progression of SLE and is expected to serve as a diagnostic marker for SLE, improving its diagnosis and treatment. Therefore, further development of non-invasive, user-friendly, highly sensitive, and specific diagnostic or detection methods for SLE, by combining clinical symptoms, signs, and laboratory parameters, will facilitate early detection, diagnosis, and treatment of SLE. Summary of the Invention

[0007] The present invention aims to address the deficiencies of the prior art and provide a use of 5-hydroxymethylcytosine. The present invention can improve the sensitivity and / or specificity of SLE diagnosis. The technical problems to be solved are not limited to the technical subject matter described herein. Other technical subjects not described herein will be clearly understood by those skilled in the art through the following description.

[0008] To solve the above technical problems, the present invention first provides any of the following applications of 5-hydroxymethylcytosine:

[0009] A1) Use in the diagnosis of systemic lupus erythematosus or in the preparation of a product for the diagnosis of systemic lupus erythematosus;

[0010] A2) Use in screening for systemic lupus erythematosus or in the preparation of a product for screening for systemic lupus erythematosus;

[0011] A3) Use in the prognosis assessment of systemic lupus erythematosus or in the preparation of a product for the prognosis assessment of systemic lupus erythematosus.

[0012] Furthermore, there is a significant difference in the modification level of 5-hydroxymethylcytosine in the systemic lupus erythematosus samples (plasma samples from systemic lupus erythematosus patients) and the control samples (plasma samples from healthy subjects).

[0013] In the above application, the substance is a reagent for detecting the 5hmC content (i.e., the 5hmC modification level in plasma cfDNA) (such as a reagent for detecting the 5hmC modification level in plasma cfDNA).

[0014] In the above application, the modification level of 5-hydroxymethylcytosine is detected by 5hmC-Seal method and hMeDIP-PCR method.

[0015] Furthermore, the molecular structure of 5hmC is:

[0016]

[0017] Furthermore, the hydroxymethylation biomarker is used in the preparation of a diagnostic kit or diagnostic equipment for systemic lupus erythematosus.

[0018] The present invention also provides a kit comprising a reagent for detecting the 5hmC modification level of a hydroxymethylation biomarker, wherein the kit has at least one of the following uses:

[0019] B1) Use in the diagnosis of systemic lupus erythematosus or in the preparation of a product for the diagnosis of systemic lupus erythematosus;

[0020] B2) Use in screening for systemic lupus erythematosus or in the preparation of a product for screening for systemic lupus erythematosus;

[0021] B3) Use in the prognosis assessment of systemic lupus erythematosus or in the preparation of a product for the prognosis assessment of systemic lupus erythematosus.

[0022] In the above-mentioned kit, the detection sample of the kit is plasma.

[0023] Furthermore, the kit includes a reagent for quantitatively detecting the level of 5hmC modification in plasma cfDNA.

[0024] Furthermore, the kit also includes reagents for extracting and purifying cfDNA in plasma.

[0025] Furthermore, the kit is a systemic lupus erythematosus diagnostic kit, which also includes a control sample, and the control sample is healthy human plasma cfDNA.

[0026] Herein, the product is a reagent, a kit or a chip.

[0027] The present invention also provides the use of the hydroxymethylation biomarker as a target in the preparation of a drug for treating or preventing systemic lupus erythematosus.

[0028] The present invention also provides a method for detecting 5-hydroxymethylcytosine, comprising the following steps:

[0029] (1) Obtaining cell-free DNA samples from patients;

[0030] (2) enriching hydroxymethylated DNA in the sample;

[0031] (3) quantitatively enriching nucleic acids in the sample that map to each of a plurality of selected loci in a target hydroxymethylation profile, wherein each of the selected loci comprises a hydroxymethylation biomarker, including the ETS1 gene;

[0032] (4) comparing the hydroxymethylation level of the patient sample with the hydroxymethylation level of the healthy sample at one or more loci of the ETS1 gene to determine the difference in hydroxymethylation level between the patient sample and the healthy sample for each hydroxymethylation biomarker to determine the modification level of 5-hydroxymethylcytosine.

[0033] Furthermore, the hydroxymethylation biomarker comprises one or more loci in a gene.

[0034] Furthermore, the sample is a plasma sample.

[0035] Furthermore, the hydroxymethylation biomarker is used to detect the modification level of 5hmC in the plasma cfDNA of the patient to be tested, and whether the patient to be tested is a systemic lupus erythematosus patient is determined based on the ratio of the modification level to the healthy sample.

[0036] Furthermore, the detection is to detect the modification level of 5hmC in different gene elements in plasma cfDNA by 5hmC-Seal method.

[0037] The present invention also provides a 5hmC-Seal detection method for determining the 5hmC modification level in plasma cfDNA.

[0038] In one embodiment of the present invention, the 5hmC-Seal method comprises the following steps:

[0039] (1) Extract cfDNA from plasma; (2) Repair and fill the ends of cfDNA fragments; (3) Connect the DNA with the filled ends to the sequencing adapter to obtain a ligation product; (4) Transfer the modification group of the sugar UDP-6-N3-glucose containing an azide modification group to the hydroxymethyl group of 5-hydroxymethylcytosine in the ligation product by T4-β-glucose transferase; (5) Add a molecule of biotin diphenylcyclooctyne-tetraethylene glycol-biotin DBCO-PEG4-Biotin to the azide-labeled 5hmC; (6) Connect the DNA containing 5hmC to the sequencing adapter. The labeled DNA fragments are bound to the solid phase material streptavidin immunomagnetic beads; (7) the solid phase material is washed multiple times with a buffer solution to remove unbound DNA fragments; (8) PCR amplification is performed using the DNA bound to the streptavidin immunomagnetic beads as a template to prepare a sequencing library, and the preparation of the sequencing library includes multiple purification steps, which are purified using magnetic beads; (9) the sequencing library is quality checked; (10) the sequencing libraries containing different barcodes are mixed at the same molar concentration, and sequencing is performed on a second-generation sequencing instrument using a standard method to obtain sequencing results.

[0040] The purpose of the above-mentioned applications and methods may be disease diagnosis, disease prognosis and / or disease treatment, or their purpose may be non-disease diagnosis, non-disease prognosis and non-disease treatment; their direct purpose may be to obtain information on intermediate results of disease diagnosis, disease prognosis and / or disease treatment, or their direct purpose may be non-disease diagnosis, non-disease prognosis and / or non-disease treatment.

[0041] After extensive and in-depth research, the inventors unexpectedly discovered that there were significant differences in the 5hmC modification levels of cfDNA between systemic lupus erythematosus samples and healthy samples. Based on this, they developed a hydroxymethylation biomarker and kit for the diagnosis and detection of systemic lupus erythematosus, which can be applied to non-invasive (or non-invasive, either) screening, diagnosis and prognostic evaluation of systemic lupus erythematosus.

[0042] The present invention proposes a use of 5-hydroxymethylcytosine. The present invention finds that hyper-5hmC (high methylation) modification peaks in cfDNA of systemic lupus erythematosus patients are related to the occurrence and development of systemic lupus erythematosus. Combined with pathway enrichment analysis and CytoHubba analysis screening, one or more related loci of 7 genes are obtained, which can be preliminarily used to diagnose SLE disease. The present invention includes new SLE patients and healthy controls, constructs an ROC curve, and screens and verifies again to determine that one or more related loci of ETS1 can be used as a biomarker for the diagnosis of systemic lupus erythematosus disease. The ratio of the 5hmC modification level in the cfDNA of the patient to be tested to the modification level of the healthy sample is used to determine whether the patient is a systemic lupus erythematosus patient. It can be applied to the development of SLE-related detection kits, thereby assisting clinicians in the early diagnosis of systemic lupus erythematosus patients.

[0043] The beneficial effects of the present invention are that the kit for diagnosing patients with systemic lupus erythematosus of the present invention, including the above-mentioned hydroxymethylation biomarker for diagnosing patients with systemic lupus erythematosus, can provide patients with systemic lupus erythematosus with a more reliable and sensitive detection basis, which helps medical staff to better diagnose patients with systemic lupus erythematosus; the substances, kits and detection methods of the hydroxymethylation biomarkers for the diagnosis and detection of systemic lupus erythematosus of the present invention have the characteristics of strong sensitivity and high specificity; and the AUC of the hydroxymethylation biomarker in the plasma cfDNA of the present invention is 0.877, and the sensitivity and specificity are 0.87 and 0.82, respectively; it can be seen that the hydroxymethylation biomarker of the present invention has high sensitivity and specificity, which helps to achieve early diagnosis and early intervention of systemic lupus erythematosus, and reduce the mortality rate of patients with systemic lupus erythematosus. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a flow chart for screening 5-hydroxymethylcytosine in SLE patients provided by the present invention. DETAILED DESCRIPTION

[0045] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the technical solutions provided by the present invention will be described in detail below in conjunction with specific embodiments, and accompanying drawings will be provided. It should be understood that the following specific embodiments are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present invention.

[0046] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0047] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0048] In order to enable those skilled in the art to better understand the present invention, see Figure 1 The following examples are provided to further illustrate the present invention.

[0049] Screening of 5-hydroxymethylcytosine:

[0050] Example 1: Obtaining 5hmC modification profiles of plasma cfDNA from SLE patients using the 5hmC-Seal method

[0051] S1: Twenty-five SLE patients were recruited from the First Affiliated Hospital of Wenzhou Medical University and divided into two stable and active groups based on their SLEDAI scores. Thirty-two healthy controls were divided into two groups (HCs). The plasma of each group of patients was mixed to form a pool, and 10 ng of plasma cfDNA was extracted from each group. Extraction can be performed by any method suitable for extracting plasma cfDNA known to those skilled in the art.

[0052] S2: First, the extracted cfDNA is tested for fragment length, then the screened cfDNA is end-repaired and filled, and the end-filled DNA is connected to the sequencing adapter to obtain the connection product. The specific method is as follows:

[0053] According to the Vazyme DNA Library Prep Kit instructions, a system containing 10 ng of cfDNA, 15 μL of End-prep mix 4, 1 μL of spike-in, and enzyme-free water to a total volume of 50 μL was prepared in a PCR tube and incubated at 20°C for 30 minutes and then at 65°C for 15 minutes. To the reaction mixture, 25 μL of Rapid Ligation buffer 2, 5 μL of Rapid DNA Ligase, 1 μL of adapter, and enzyme-free water to a total volume of 100 μL were added, incubated at 20°C for 15 minutes, and then maintained at 4°C. The reaction product was purified using AmpureXP beads and eluted with 21 μL of enzyme-free water to obtain the final DNA ligation sample.

[0054] S3: Through T4-β-glucose transferase, the modification group of the sugar UDP-6-N3-glucose containing an azide modification group is transferred to the hydroxymethyl group of the ligation product 5hmC.

[0055] S4: Click chemistry was used to add a molecule of biotin diphenylcyclooctyne-tetraethylene glycol-biotin DBCO-PEG4-Biotin to the azide-labeled 5hmC. The specific method is as follows:

[0056] A total volume of 25 μL of a labeling reaction mixture (including T4-β-glucose transferase, UDP-6-N3-glucose with an azide-modified group, 10× buffer, and 21 μL of the purified product) was prepared and incubated at 37°C for 2 hours. 2.5 μL of diphenylcyclooctyne-tetraethylene glycol-biotin was added to the reaction product, and the mixture was incubated at 37°C for 2 hours. 10 μg of sheared salmon sperm DNA was added to the reaction mixture, and the reaction mixture was purified using a Bio-Rad Micro Bio-spin 30 column, and the purified product was brought to a volume of 50 μL.

[0057] S5: The DNA fragments containing the 5hmC marker are bound to the solid phase material streptavidin immunomagnetic beads through solid phase affinity reaction.

[0058] S6: Unbound DNA fragments were removed by multiple washings using a buffer containing Tris-HCl, EDTA, NaCl, and the surfactant Tween 20.

[0059] S7: DNA bound to streptavidin immunomagnetic beads is used as a template for PCR amplification to prepare a sequencing library; the preparation of the sequencing library includes multiple purification steps, which are performed using magnetic beads.

[0060] S8: Perform quality check on the sequencing library.

[0061] S9: Sequencing libraries that have passed quality inspection can be used for high-throughput sequencing. Libraries containing a certain number of different barcodes are mixed at the same molar concentration and sequenced using standard methods on a second-generation sequencer to obtain sequencing results.

[0062] Example 2: Determination of differentially modified regions using bioinformatics analysis

[0063] The fragment size and enrichment level of sequencing were calculated using MACS2 with default parameters. Peak Annotator can confirm 5hmC enrichment on genes. The modified fragment counts in each Ensembl gene region obtained using BEDTools were used to calculate the FPKM of 5hmC. The distribution of 5hmC modifications on different gene elements such as 5'UTR, CDS, 3'UTR, and the distance between the modified peak and the transcription start site were further analyzed. HOMER (version 4.4) was used to analyze the motifs near the 5hmC peak. In all samples, peaks with enrichment multiples greater than 2 and q values less than 1e-6 were defined as 5hmC enrichment regions. PePr v1.1.18 was used to identify differentially modified gene regions between SLE and healthy samples, and those with fold changes (Fc) greater than 1.5 and p values less than 0.005 were selected for downstream analysis.

[0064] Example 3: Screening for 5-hydroxymethylcytosine in SLE

[0065] To explore the biological functions of abnormally modified 5hmC regions, functional enrichment annotation analysis was performed using the KOBAS database, which includes the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and the National Human Genome Research Institute (NHGRI). SLE-associated genes were selected from the DisGeNET database. A classifier was used to identify the top 10 genes containing one or more associated loci in the central gene network. Biomarkers relevant to SLE diagnosis were further screened by taking the intersection. To further screen for hydroxymethylation biomarkers for SLE, the present invention expanded the sample size for revalidation. In the validation cohort, 5hmC modification levels at one or more ETS1-associated loci were significantly increased in cfDNA from SLE patients compared to healthy controls, further confirming that one or more ETS1-associated loci can be used to diagnose SLE.

[0066] Evaluation of the diagnostic performance of 5-hydroxymethylcytosine:

[0067] In this study, the receiver operating characteristic curve (ROC) was used to evaluate the diagnostic efficacy of one or more related loci of ETS1. The area under the ROC curve (AUC) can reflect the ability of a diagnostic index to distinguish between positive and negative diagnoses. The larger the AUC value, the higher the diagnostic accuracy. Generally, when 0.5 < AUC < 0.7, the diagnostic value is low; when 0.7 < AUC < 0.9, the diagnostic value is medium; when AUC ≥ 0.9, the diagnostic value is high.

[0068] The results showed that the AUC of the hydroxymethylation biomarker of the present invention for identifying SLE was 0.877, and the sensitivity and specificity were 0.87 and 0.82, respectively. The above results indicate that one or more related loci of ETS1 can be used as biomarkers for SLE diagnosis.

[0069] Example 4: Construction of a diagnostic kit based on 5-hydroxymethylcytosine

[0070] By analyzing and comparing the hydroxymethylation biomarker modification levels in samples from SLE patients and healthy individuals, it was found that the ratio of the 5hmC modification levels of the above hydroxymethylation biomarker in SLE patients and healthy individuals can be used as a biomarker for diagnosing SLE patients, providing a strong reference basis for kit preparation. If the 5hmC modification level of one or more related loci of ETS1 in the cfDNA of a sample is increased by 5.9 times or more compared to the 5hmC modification level of healthy individuals, then the sample is determined to be a systemic lupus erythematosus sample. This kit can be used to assist clinicians in the early diagnosis of SLE patients without typical symptoms and suspected SLE patients. This simple diagnostic design enables point-of-care testing in hospitals.

[0071] The above has described the embodiments of the present invention in detail, but the present invention is not limited thereto, within the knowledge scope of those skilled in the art. Therefore, any modifications, equivalent replacements, improvements, etc. made without departing from the spirit and scope of the present invention shall be included within the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modification examples falling within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries; these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A use of 5-hydroxymethylcytosine, characterized in that: Include one of the following applications: A1) Use in the preparation of a product for the diagnosis of systemic lupus erythematosus; A2) Use in the preparation of a product for screening of systemic lupus erythematosus; A3) Use in the preparation of a product for prognostic assessment of systemic lupus erythematosus; The modification level of 5-hydroxymethylcytosine was detected by 5hmC-Seal method and hMeDIP-PCR method; The modification level of 5-hydroxymethylcytosine was detected by the following method: (1) Obtain cfDNA samples from patients; (2) enriching hydroxymethylated DNA in the sample; (3) quantitatively enriching nucleic acids in the sample that map to each of a plurality of selected loci in a target hydroxymethylation profile, wherein each of the selected loci comprises a hydroxymethylation biomarker, including the ETS1 gene; (4) comparing the hydroxymethylation level of the patient sample with the hydroxymethylation level of the healthy sample at one or more loci of the ETS1 gene to determine the difference in hydroxymethylation level between the patient sample and the healthy sample for each hydroxymethylation biomarker to determine the modification level of 5-hydroxymethylcytosine.

2. The use of 5-hydroxymethylcytosine according to claim 1, characterized in that The hydroxymethylation biomarkers comprise one or more loci in a gene.

Images