Methylation region marker combination for early auxiliary diagnosis of gestational diabetes mellitus and use thereof

Abstract

Disclosed are a methylation region marker combination for early auxiliary diagnosis of gestational diabetes mellitus and a use thereof. The methylation region marker combination is a combination of seven methylation regions related to early gestational diabetes mellitus, such as chr11:118498190-118498259, chr13:111841565-111842079, chr14:104208350-104208437, chr1:205089780-205089897, chr20:43996165-43996235, chr22:48960889-48961073, and chr3:48697595-48697883. A specific amplification primer for a methylation region marker can be used for preparing an early auxiliary diagnosis kit for gestational diabetes mellitus.

Description

A methylation region marker combination for early auxiliary diagnosis of gestational diabetes and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on November 3, 2023, with application number 202311450861.5 and invention name “A combination of methylation region markers for early auxiliary diagnosis of gestational diabetes and its application”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of genetic engineering and women's reproductive health, and relates to a methylation region marker for early auxiliary diagnosis of gestational diabetes and its application. Background Art

[0004] Gestational diabetes mellitus (GDM) is a common pregnancy disorder, specifically impaired glucose tolerance first diagnosed during pregnancy. Studies have shown that GDM, a high-risk pregnancy, increases the risk of gestational hypertension and preeclampsia. Pregnant women with GDM have a risk of developing type 2 diabetes that is over seven times higher than healthy women. Furthermore, GDM can negatively impact both the short-term and long-term growth and development of offspring. In the short term, it can lead to neonatal hypoglycemia, hyperbilirubinemia, and macrosomia. In the long term, it increases the risk of obesity, type 2 diabetes, hypertension, and other metabolic syndromes in offspring, placing a severe psychological and economic burden on families and society.

[0005] In recent years, a growing number of studies have focused on the role of epigenetic markers in the development and progression of GDM. Epigenetics, because it integrates information from both genetic factors (such as gene mutations) and non-genetic factors (such as environmental influences and lifestyle changes), has been recognized in recent years as having a stronger and more direct impact on revealing disease risk. DNA methylation, as one of the most important forms of epigenetic inheritance, is directly or indirectly involved in the regulation of gene expression. Early studies of candidate genes in the field of GDM have shown that methylation levels in certain key genes alter the pathological process of GDM. For example, abnormal methylation in the promoter regions of the classic adiponectin (ADIPOQ) and leptin genes is significantly associated with maternal glucose metabolism. With the rapid development of biotechnology, whole-genome methylation testing based on microarray or sequencing technologies has been widely applied in GDM-related research, identifying more potential methylation sites. The most representative of these is the Global Pregnancy and Childhood Epigenetics Consortium, whose research subjects all used the Illumina 450K array for genome-wide methylation profiling. This multifaceted assessment, combined with phenotypes such as maternal smoking, BMI, hypertension, and newborn weight, identified numerous methylation sites associated with various pregnancy states, including GDM. Another study from Taiwan, China, showed that genome-wide methylation profiling of peripheral blood from women with GDM and healthy women before delivery using a methylation array revealed over 200 differentially methylated sites enriched in pathways such as endocrine disorders, metabolic diseases, carbohydrate metabolism, and lipid metabolism.

[0006] In summary, although genome-wide methylation studies have greatly improved the identification of GDM-associated methylation sites, most of these studies have explored changes in methylation profiles after the onset of GDM and lack evidence from prospective cohort studies. The methylation sites identified in existing studies do not adequately explain the etiology of GDM. Currently, there are no reports on the application of methylation for the early diagnosis of gestational diabetes. If methylation profiles associated with early gestational diabetes and genes associated with early gestational diabetes affected by methylation can be identified, and corresponding diagnostic kits can be developed, this would significantly advance the diagnosis of early gestational diabetes in my country and open up new avenues for drug screening and efficacy evaluation.

[0007] Summary of the Invention

[0008] The primary purpose of this application is to address the above-mentioned problems and propose a methylation region combination marker for early auxiliary diagnosis of gestational diabetes.

[0009] The second purpose of the present application is to provide the above-mentioned specific primer combination for amplifying the above-mentioned methylated region.

[0010] The third object of the present application is to provide the use of the above-mentioned specific primer combination in the preparation of an early auxiliary diagnosis kit for gestational diabetes.

[0011] The fourth purpose of this application is to provide an early auxiliary diagnosis kit for gestational diabetes.

[0012] By studying the methylation data of patients with early gestational diabetes and healthy controls, the inventors searched for highly specific and sensitive methylation region combinations that are highly correlated with the early risk of gestational diabetes, and developed an early auxiliary diagnosis kit for gestational diabetes that can be used in clinical applications. This provides data support for the early auxiliary diagnosis of gestational diabetes and the discovery of new small molecule drugs with potential therapeutic value.

[0013] The purpose of this application is achieved through the following technical solutions:

[0014] A methylation region marker for early auxiliary diagnosis of gestational diabetes. The methylation region marker combination is a methylation region marker associated with early gestational diabetes.

[0015] chr11:118498190-118498259, chr13:111841565-111842079,

[0016] The combination of chr14:104208350-104208437, chr1:205089780-205089897, chr20:43996165-43996235, chr22:48960889-48961073, chr3:48697595-48697883.

[0017] The specific amplification primers for the methylated region markers are as follows (the base R represents A / G):

[0018] The specific amplification primers for chr11:118498190-118498259 are: SEQ ID NO.1, SEQ ID NO.2;

[0019] The specific amplification primers for chr13:111841565-111842079 are: SEQ ID NO.3, SEQ ID NO.4;

[0020] The specific amplification primers for chr14:104208350-104208437 are: SEQ ID NO.5, SEQ ID NO.6;

[0021] The specific amplification primers for chr1:205089780-205089897 are: SEQ ID NO.7, SEQ ID NO.8;

[0022] The specific amplification primers for chr20:43996165-43996235 are: SEQ ID NO.9, SEQ ID NO.10;

[0023] The specific amplification primers for chr22:48960889-48961073 are: SEQ ID NO.11, SEQ ID NO.12;

[0024] The specific amplification primers for chr3:48697595-48697883 are: SEQ ID NO.13, SEQ ID NO.14.

[0025] The use of the methylation region marker in the preparation of an early auxiliary diagnosis kit for gestational diabetes mellitus.

[0026] A kit for the early auxiliary diagnosis of gestational diabetes mellitus, which is used to detect the methylation degree of chr11:118498190-118498259, chr13:111841565-111842079, chr14:104208350-104208437, chr1:205089780-205089897, chr20:43996165-43996235, chr22:48960889-48961073, and chr3:48697595-48697883 in peripheral blood DNA.

[0027] The diagnostic kit contains specific amplification primers for the above-mentioned methylation region markers.

[0028] The diagnostic kit contains specific amplification primers for methylation region markers, which are (the base R represents A / G):

[0029] The specific amplification primers for chr11:118498190-118498259 are: SEQ ID NO.1, SEQ ID NO.2;

[0030] The specific amplification primers for chr13:111841565-111842079 are: SEQ ID NO.3, SEQ ID NO.4;

[0031] The specific amplification primers for chr14:104208350-104208437 are: SEQ ID NO.5, SEQ ID NO.6;

[0032] The specific amplification primers for chr1:205089780-205089897 are: SEQ ID NO.7, SEQ ID NO.8;

[0033] The specific amplification primers for chr20:43996165-43996235 are: SEQ ID NO.9, SEQ ID NO.10;

[0034] The specific amplification primers for chr22:48960889-48961073 are: SEQ ID NO.11, SEQ ID NO.12;

[0035] The specific amplification primers for chr3:48697595-48697883 are: SEQ ID NO.13, SEQ ID NO.14;

[0036] The diagnostic kit further comprises reagents used in any one or more of the following methylation detection methods, including reagents used in: whole genome bisulfite sequencing (WGBS), pyrosequencing, bisulfite sequencing, methylation-specific polymerase chain reaction (MS-PCR), bisulfite-specific polymerase chain reaction, methylation-sensitive restriction endonuclease-PCR / Southern method, combined bisulfite restriction endonuclease method (COBRA), digital polymerase chain reaction, restriction landmark genome scanning, CpG island microarray, single nucleotide primer extension SNUPE, methylation profiling, and methylation chip.

[0037] The diagnostic kit further comprises reagents for processing samples.

[0038] The treatment may include the steps of extracting DNA and converting cytosine into uracil.

[0039] The reagent used in the step of converting cytosine to uracil is most commonly a bisulfite reagent.

[0040] The bisulfite reagent includes a bisulfite buffer and a protection buffer.

[0041] The DNA extraction reagent may include a lysis buffer, a binding buffer, a washing buffer and an elution buffer.

[0042] The lysis buffer comprises a protein denaturant, a detergent, a pH buffer and a nuclease inhibitor.

[0043] The binding buffer comprises a protein denaturant and a pH buffer.

[0044] The pH buffer is selected from one or more of Tris, boric acid, phosphate, and MES.

[0045] The nuclease inhibitor is selected from one or more of EDTA, EGTA and DEPC.

[0046] The washing buffer is selected from one or more of Tris, boric acid, sorbitol, polyethylene glycol and mercaptoethanol.

[0047] The elution buffer is selected from one or more of NaCl, Tris-HCl and EDTA.

[0048] (1) Establish a unified standard specimen library and database: collect blood samples that meet the standards according to standard operating procedures (SOPs) and systematically collect complete demographic and clinical data.

[0049] (2) Bisulfite whole-genome sequencing: Peripheral blood samples from patients with gestational diabetes and healthy controls in early pregnancy were selected, and bisulfite whole-genome sequencing was used to identify differentially methylated regions between the two groups across the entire genome.

[0050] (3) Development of an early auxiliary diagnosis kit for gestational diabetes: A kit for the early auxiliary diagnosis of gestational diabetes is developed based on the significantly different methylation regions screened in the peripheral blood of gestational diabetes patients and healthy controls in early pregnancy.

[0051] Specifically, the experimental methods of the study mainly include the following parts:

[0052] 1. Selection of research samples

[0053] This study involved a prospective cohort of naturally conceived women enrolled at the Nanjing Maternal and Child Health Hospital. Pregnant women with normal first-trimester glucose levels but diagnosed with GDM during a 75g oral glucose tolerance test (OGTT) in the second trimester and who subsequently delivered successfully served as the case group. Pregnant women with normal first-trimester glucose levels and who subsequently delivered successfully served as healthy controls. Pregnant women with pregnancy complications other than GDM and pre-existing diabetes were excluded.

[0054] Blood collection and retention in early pregnancy;

[0055] A total of 221 samples that met the criteria were included in this study.

[0056] Genomic DNA from blood samples was extracted using the QIAamp DNA Kit according to standard procedures. Typically, 20-50 ng / μl DNA was obtained, with a purity (UV 260OD to 280OD ratio) of 1.6-2.0.

[0057] 3. Illumina HiSeq X Ten platform whole genome methylation detection

[0058] Extraction of peripheral blood DNA: DNA was extracted using the QIAamp DNA Mini Kit.

[0059] Fragmentation: Add approximately 1% exogenous phage DNA, 1.25 μL Fragment Buffer, 2.5 μL Enzyme to 100 ng of sample DNA, and add ddH2O to make up to 12.5 μL. Heat at 4°C for 1 minute, then at 37°C for 14 minutes, and let stand at 4°C to fragment the DNA into 200-300 bp fragments.

[0060] End repair and A-tail addition: Take 12.5μL of DNA, add 1.75μL of ER-AT buffer and 0.75μL of ER-AT Enzyme, heat at 20℃ for 30min, heat at 65℃ for 30min, and let stand at 4℃.

[0061] DNA ligation: Use the KAPA DNA HyperPlus Kit. Take 15 μL of DNA, add 7.5 μL of Ligation Buffer, 2.5 μL of Enzyme, and 1 μL of Methy-Adapter. Add ddH2O to make up to 27.5 μL. Heat at 20°C for 4 hours or store at 4°C overnight.

[0062] Fragment purification: After equilibration of the DNA beads at room temperature for 0.5 h, add 0.8× the volume of the reaction mixture from the previous step to the beads and mix thoroughly. Incubate at room temperature for 5 min. Place on a magnetic rack until the liquid clarifies and discard the supernatant. Then, add 200 μL of 80% ethanol (diluted with ddH2O) to resuspend the beads. Place on a magnetic rack and discard the supernatant. Repeat this step once. Elute with EB and transfer the supernatant to a new PCR tube on a magnetic rack.

[0063] Bisulfite conversion: using EZ DNA Methylation-Lightning TMThe DNA sulfite conversion kit (D5030T) is used to convert DNA. Before use, add 24 mL of 100% ethanol to 6 mL of M-Wash Buffer Concentrate to prepare M-Wash Buffer.

[0064] PCR amplification: High-efficiency PCR reactions were performed using the KAPA HIFI HotStart ReadyMix (2802). The reaction was heated at 98°C for 45 seconds, followed by ten cycles of heating at 98°C for 15 seconds, 65°C for 30 seconds, and 72°C for 30 seconds. After the cycle, the reaction was heated at 72°C for 1 minute and allowed to stand at 4°C.

[0065] Fragment purification: After the DNA beads were equilibrated at room temperature for 0.5 h, 0.8× the volume of the reaction system in the previous step was added to the beads to complete the system and mix well. Incubate at room temperature for 5 min, place on a magnetic rack until the liquid is clear, and discard the supernatant; add 200 μL 80% ethanol (diluted with ddH2O) to resuspend the magnetic beads, place on a magnetic rack, discard the supernatant, and repeat this step once; elute with EB, transfer the supernatant on a magnetic rack to a new PCR tube, and determine the concentration.

[0066] Sequencing: The prepared WGBS library was subjected to whole-genome sequencing using the Illumina HiSeq X Ten high-throughput sequencing platform.

[0067] 4. Statistical Analysis Methods

[0068] The t-test was used to compare the methylation levels of the global and genomic regions between the case and control groups. The hypergeometric distribution test was used for enrichment analysis of differentially methylated regions.

[0069] DNA methylation differential analysis was performed using R statistical software. The bsseq package was used to smooth bisulfite sequencing data. Pairwise differential methylation analysis was performed using the DSS package. In this study, differentially methylated locus (DML) was defined as a region with a P value less than 1 × 10 -5 Differentially methylated regions (DMRs) were identified as regions with a minimum width of 50 bp and at least three CpG regions formed by combining differential regions with a P value less than 0.05. Lasso regression model dimensionality reduction was performed on all differentially hypermethylated regions based on the glmnet package of R software, and finally 7 candidate methylation regions were obtained.

[0070] To further investigate the effectiveness of the combined indication of these seven methylation regions for early diagnosis, we constructed a mathematical formula that comprehensively considers the association and strength of the methylation level of each region with the early onset of gestational diabetes. Specifically, we assigned a score to each study subject, taking into account the methylation profile of each region. The algorithm for evaluating the score is as follows: evaluation score = (18.07×chr13:111841565-111842079-16.93×chr11:118498190-118498259-5.56×chr14:104208350-104208437+4.73×chr1:205089780-205089897-0.12×chr20:43996165-43996235+8.58×chr22:48960889-48961073+6.33×chr3:48697595-48697883). The obtained score coefficient and cut-off value were directly applied to the samples of the methylation association analysis study.

[0071] Statistical analysis was performed using specialized statistical analysis software (R language, v4.0.2). The statistical significance level was set at 0.05, and all statistical tests were two-sided.

[0072] 5. Preparation of diagnostic kit

[0073] Whole-genome methylation sequencing was performed on the Illumina HiSeq X Ten platform. Methylation differential analysis identified significant differentially methylated regions in the peripheral blood of women with gestational diabetes and healthy controls during early pregnancy, serving as diagnostic indicators for the early development of gestational diabetes. Finally, the differentially methylated regions identified as associated with the early onset of gestational diabetes were used to develop a diagnostic kit (chr11:118498190-118498259, chr13:111841565-111842079, chr14:104208350-104208437, chr1:205089780-205089897, chr20:43996165-43996235, chr22:48960889-48961073, chr3:48697595-48697883). Diagnostic reagents can include specific primers for these methylated regions, as well as reagents such as DNA extraction and bisulfite conversion, all of which can be assembled in one kit or dispersed in a series of kits for combined use.

[0074] The following is further description of this application:

[0075] We used Illumina HiSeq X Ten bisulfite whole-genome methylation sequencing to obtain relevant results in peripheral blood samples from the 221 eligible patients with gestational diabetes and healthy controls during early pregnancy.

[0076] According to the Illumina HiSeq X Ten whole-genome methylation assay, the applicant detected seven differentially methylated regions in the peripheral blood of the "gestational diabetes mellitus" group and the "healthy control" group, including chr11:118498190-118498259, chr13:111841565-111842079, chr14:104208350-104208437, chr1:205089780-205089897, chr20:43996165-43996235, chr22:48960889-48961073, and chr3:48697595-48697883.

[0077] The combination of 7 differentially methylated regions was used for the early diagnosis of gestational diabetes, and it was found that the combination could well distinguish cases from controls.

[0078] Based on the above experimental results, the applicant prepared a kit for early auxiliary diagnosis of gestational diabetes, which contains specific primers and other detection reagents for measuring the above methylation regions in the DNA of the subject's blood sample.

[0079] Specifically, the combination of these seven methylation regions, or the related diagnostic kit composed of specific primers of these seven methylation regions, can help in the early auxiliary diagnosis of gestational diabetes, and provide support for clinicians to assist in judging the potential disease status of pregnant women and adopt more personalized prevention and treatment plans in a timely manner.

[0080] The advantages of the methylation region provided in this application for early auxiliary diagnosis of gestational diabetes and its application are:

[0081] (1) DNA methylation is a new biomarker that, unlike traditional biomarkers, can be detected in the early stages of pathology or sub-health, greatly improving the sensitivity and specificity of disease diagnosis. The discovery of early methylation characteristics in people with gestational diabetes mellitus can help clinicians provide precise intervention quickly and accurately, and adopt more personalized prevention and treatment plans for high-risk groups in a timely manner.

[0082] (2) The methylation kit is a systematic and comprehensive diagnostic kit that helps reflect the methylation levels of different subjects. Combined with the subject's metabolomics data, it can quickly determine the subject's early onset risk, which is a powerful supplement to the previous prediction of gestational diabetes.

[0083] (3) Using a rigorous validation and evaluation system, the inventors used bisulfite whole-genome sequencing technology to directly measure methylated DNA sequences and further analyze and obtain disease-related methylation maps. The application of the above methods and strategies has accelerated and ensured the clinical application of DNA methylation and diagnostic kits, and also provided a reference for the development of methods and strategies for other disease biomarkers.

[0084] This application studies the methylation data of peripheral blood from early pregnancy in patients with gestational diabetes and healthy controls to identify methylation-related markers that can be used for early detection of gestational diabetes. Therefore, this application obtains a methylation database for early gestational diabetes and related genes affected by methylation. By applying this data to differentially methylated regions in early gestational diabetes, this application promotes the clinical translation of gestational diabetes-related research results and the comparison of research results between different laboratories, providing basic data and scientific basis for early screening and precise intervention for high-risk populations, and assisting in the discovery of new small molecule drug targets with potential therapeutic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1: ROC curve showing genome-wide methylation association study in peripheral blood of patients with gestational diabetes and healthy controls during early pregnancy. DETAILED DESCRIPTION

[0086] The present application is further described below through examples.

[0087] Example 1

[0088] Selection of research samples

[0089] This study involved a prospective cohort of naturally conceived women enrolled at the Nanjing Maternal and Child Health Hospital. Pregnant women with normal first-trimester glucose levels but diagnosed with GDM during a 75g oral glucose tolerance test (OGTT) in the second trimester and who subsequently delivered successfully served as the case group. Pregnant women with normal first-trimester glucose levels and who subsequently delivered successfully served as healthy controls. Pregnant women with pregnancy complications other than GDM and pre-existing diabetes were excluded.

[0090] Blood collection and retention in early pregnancy;

[0091] A total of 221 samples that met the criteria were included in this study.

[0092] Genomic DNA from blood samples was extracted using the QIAamp DNA Kit according to standard procedures. Typically, 20-50 ng / μl DNA was obtained, with a purity (UV 260OD to 280OD ratio) of 1.6-2.0.

[0093] 3. Illumina HiSeq X Ten platform whole genome methylation detection

[0094] Extraction of peripheral blood DNA: DNA was extracted using the QIAamp DNA Mini Kit.

[0095] Fragmentation: Add approximately 1% exogenous phage DNA, 1.25 μL Fragment Buffer, 2.5 μL Enzyme to 100 ng of sample DNA, and add ddH2O to make up to 12.5 μL. Heat at 4°C for 1 minute, then at 37°C for 14 minutes, and let stand at 4°C to fragment the DNA into 200-300 bp fragments.

[0096] End repair and A-tail addition: Take 12.5μL of DNA, add 1.75μL of ER-AT buffer and 0.75μL of ER-AT Enzyme, heat at 20℃ for 30min, heat at 65℃ for 30min, and let stand at 4℃.

[0097] DNA ligation: Use the KAPA DNA HyperPlus Kit. Take 15 μL of DNA, add 7.5 μL of Ligation Buffer, 2.5 μL of Enzyme, and 1 μL of Methy-Adapter. Add ddH2O to make up to 27.5 μL. Heat at 20°C for 4 hours or store at 4°C overnight.

[0098] Fragment purification: After equilibration of the DNA beads at room temperature for 0.5 h, add 0.8× the volume of the reaction mixture from the previous step to the beads and mix thoroughly. Incubate at room temperature for 5 min. Place on a magnetic rack until the liquid clarifies and discard the supernatant. Then, add 200 μL of 80% ethanol (diluted with ddH2O) to resuspend the beads. Place on a magnetic rack and discard the supernatant. Repeat this step once. Elute with EB and transfer the supernatant to a new PCR tube on a magnetic rack.

[0099] Bisulfite conversion: using EZ DNA Methylation-Lightning TM The DNA sulfite conversion kit (D5030T) is used to convert DNA. Before use, add 24 mL of 100% ethanol to 6 mL of M-Wash Buffer Concentrate to prepare M-Wash Buffer.

[0100] PCR amplification: High-efficiency PCR reactions were performed using the KAPA HIFI HotStart ReadyMix (2802). The reaction was heated at 98°C for 45 seconds, followed by ten cycles of heating at 98°C for 15 seconds, 65°C for 30 seconds, and 72°C for 30 seconds. After the cycle, the reaction was heated at 72°C for 1 minute and allowed to stand at 4°C.

[0101] Fragment purification: After the DNA beads were equilibrated at room temperature for 0.5 h, 0.8× the volume of the reaction system in the previous step was added to the beads to complete the system and mix well. Incubate at room temperature for 5 min, place on a magnetic rack until the liquid is clear, and discard the supernatant; add 200 μL 80% ethanol (diluted with ddH2O) to resuspend the magnetic beads, place on a magnetic rack, discard the supernatant, and repeat this step once; elute with EB, transfer the supernatant on a magnetic rack to a new PCR tube, and determine the concentration.

[0102] Sequencing: The prepared WGBS library was subjected to whole-genome sequencing using the Illumina HiSeq X Ten high-throughput sequencing platform.

[0103] 4. Statistical Analysis Methods

[0104] The t-test was used to compare the methylation levels of the global and genomic regions between the case and control groups. The hypergeometric distribution test was used for enrichment analysis of differentially methylated regions.

[0105] DNA methylation differential analysis was performed using R statistical software. The bsseq package was used to smooth bisulfite sequencing data. Pairwise differential methylation analysis was performed using the DSS package. In this study, differentially methylated locus (DML) was defined as a region with a P value less than 1 × 10 -5 Differentially methylated regions (DMRs) were defined as regions with a minimum width of 50 bp and at least three CpG regions that were combined with differential regions with a P value less than 0.05.

[0106] To further investigate the effectiveness of the combined indication of these seven methylation regions for early diagnosis, we constructed a mathematical formula that comprehensively considers the association and strength of the methylation level of each region with the early onset of gestational diabetes. Specifically, we assigned a score to each study subject, taking into account the methylation profile of each region. The algorithm for evaluating the score is as follows: evaluation score = (18.07×chr13:111841565-111842079-16.93×chr11:118498190-118498259-5.56×chr14:104208350-104208437+4.73×chr1:205089780-205089897-0.12×chr20:43996165-43996235+8.58×chr22:48960889-48961073+6.33×chr3:48697595-48697883). The obtained score coefficient and cut-off value were directly applied to the samples of the methylation association analysis study.

[0107] Statistical analysis was performed using specialized statistical analysis software (R language, v4.0.2). The statistical significance level was set at 0.05, and all statistical tests were two-sided.

[0108] 5. Result analysis:

[0109] To evaluate the predictive efficacy of this model for early gestational diabetes, R software was used to construct and evaluate the seven methylation regions described above. A logistic regression model was constructed using early gestational diabetes as the phenotype. Data from 221 whole-genome methylation sequencing samples were used, 80% of which were used for model training and 20% for validation. The rROC software package was used to plot the receiver operating characteristic (ROC) curve, also known as the sensitivity curve. The curve plots the false positive rate on the horizontal axis and the true positive rate on the vertical axis. The area under the curve (AUC) represents the model's ability to distinguish patients with gestational diabetes from healthy controls. A value greater than 0.85 indicates excellent classification performance. The model achieved an AUC of 0.874 in the validation set, indicating good discrimination between patients with gestational diabetes and healthy controls.

[0110] Example 2: Preparation of diagnostic kit

[0111] Whole-genome methylation sequencing was performed on the Illumina HiSeq X Ten platform. Methylation differential analysis identified significant differentially methylated regions in the peripheral blood of women with gestational diabetes and healthy controls during early pregnancy as indicators for the early diagnosis of gestational diabetes. Finally, the differentially methylated regions identified as associated with the early onset of gestational diabetes were used to develop a diagnostic kit (chr11:118498190-118498259, chr13:111841565-111842079, chr14:104208350-104208437, chr1:205089780-205089897, chr20:43996165-43996235, chr22:48960889-48961073, chr3:48697595-48697883).

[0112] Diagnostic reagents can include specific primers for these methylated regions, as well as reagents such as DNA extraction and bisulfite conversion, all of which can be assembled in one kit or dispersed in a series of kits for combined use.

[0113] Specifically, a kit for the early diagnosis of gestational diabetes mellitus is provided. The kit is used to detect the methylation degree of chr11:118498190-118498259, chr13:111841565-111842079, chr14:104208350-104208437, chr1:205089780-205089897, chr20:43996165-43996235, chr22:48960889-48961073, and chr3:48697595-48697883 in peripheral blood DNA. The kit contains the following specific amplification primer combinations (the base R represents A / G):

[0114] The kit also contains reagents for processing samples, wherein the processing includes the steps of extracting DNA and converting cytosine into uracil; wherein:

[0115] The reagent used in the step of converting cytosine into uracil is most commonly a bisulfite reagent, including a bisulfite buffer and a protective buffer;

[0116] The DNA extraction reagents include a lysis buffer, a binding buffer, a washing buffer and an elution buffer, wherein the lysis buffer includes a protein denaturant, a detergent, a pH buffer and a nuclease inhibitor; the binding buffer includes a protein denaturant and a pH buffer; the pH buffer includes one or more of Tris, boric acid, phosphate and MES; the nuclease inhibitor includes one or more of EDTA, EGTA and DEPC; the washing buffer includes one or more of Tris, boric acid, sorbitol, polyethylene glycol and mercaptoethanol; and the elution buffer includes one or more of NaCl, Tris-HCl and EDTA.

[0117] The kit also includes reagents used in any one or more methylation detection methods, including: whole genome bisulfite sequencing (WGBS), pyrosequencing, bisulfite sequencing, methylation-specific polymerase chain reaction (MS-PCR), bisulfite-specific polymerase chain reaction, methylation-sensitive restriction endonuclease-PCR / Southern method, combined bisulfite restriction endonuclease method (COBRA), digital polymerase chain reaction, restriction landmark genome scanning, CpG island microarray, single nucleotide primer extension SNUPE, methylation profiling, and methylation chip.

Claims

1. A methylation region marker combination for early auxiliary diagnosis of gestational diabetes, characterized in that: This methylation region marker combination is a combination of methylation regions associated with early gestational diabetes chr11:118498190-118498259, chr13:111841565-111842079, chr14:104208350-104208437, chr1:205089780-205089897, chr20:43996165-43996235, chr22:48960889-48961073, and chr3:48697595-48697883.

2. A specific amplification primer combination for amplifying the methylation region marker according to claim 1, characterized in that: The primer combination is: The specific amplification primers for chr11:118498190-118498259 are: SEQ ID NO.1, SEQ ID NO.2; The specific amplification primers for chr13:111841565-111842079 are: SEQ ID NO.3, SEQ ID NO.4; The specific amplification primers for chr14:104208350-104208437 are: SEQ ID NO.5, SEQ ID NO.6; The specific amplification primers for chr1:205089780-205089897 are: SEQ ID NO.7, SEQ ID NO.8; The specific amplification primers for chr20:43996165-43996235 are: SEQ ID NO.9, SEQ ID NO.10; The specific amplification primers for chr22:48960889-48961073 are: SEQ ID NO.11, SEQ ID NO.12; The specific amplification primers for chr3:48697595-48697883 are: SEQ ID NO.13, SEQ ID NO.

14.

3. Use of the specific amplification primer combination according to claim 2 in the preparation of an early auxiliary diagnosis kit for gestational diabetes mellitus.

4. A kit for early auxiliary diagnosis of gestational diabetes, characterized in that: The kit contains reagents for detecting the methylation degree of chr11:118498190-118498259, chr13:111841565-111842079, chr14:104208350-104208437, chr1:205089780-205089897, chr20:43996165-43996235, chr22:48960889-48961073, and chr3:48697595-48697883 in peripheral blood DNA.

5. The auxiliary diagnostic kit according to claim 4, characterized in that: The kit contains the specific amplification primer combination described in claim 2.

6. The auxiliary diagnostic kit according to claim 5, characterized in that: The kit also contains reagents for processing the samples.

7. The auxiliary diagnostic kit according to claim 6, characterized in that: The treatment comprises the steps of extracting DNA and converting cytosine into uracil; wherein: The reagent used in the step of converting cytosine into uracil is most commonly a bisulfite reagent, including a bisulfite buffer and a protective buffer; The DNA extraction reagent comprises a lysis buffer, a binding buffer, a washing buffer and an elution buffer, wherein the lysis buffer comprises a protein denaturant, a detergent, a pH buffer and a nuclease inhibitor; the binding buffer comprises a protein denaturant and a pH buffer; the pH buffer is selected from one or more of Tris, boric acid, phosphate and MES; the nuclease inhibitor is selected from one or more of EDTA, EGTA or DEPC; the washing buffer is selected from one or more of Tris, boric acid, sorbitol, polyethylene glycol and mercaptoethanol; and the elution buffer is selected from one or more of NaCl, Tris-HCl or EDTA.

8. The auxiliary diagnostic kit according to claim 5, characterized in that: The kit also includes any one or more reagents used in the methylation detection method.

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