CN117947169A - Methylation gene marker combination for early detection of endometrial cancer, multiplex PCR detection kit and application thereof - Google Patents

Methylation gene marker combination for early detection of endometrial cancer, multiplex PCR detection kit and application thereof Download PDF

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CN117947169A
CN117947169A CN202410149219.1A CN202410149219A CN117947169A CN 117947169 A CN117947169 A CN 117947169A CN 202410149219 A CN202410149219 A CN 202410149219A CN 117947169 A CN117947169 A CN 117947169A
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贾卫华
廖颖
杜艳
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Sun Yat Sen University Cancer Center
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Abstract

The invention belongs to the technical field of biology, and particularly relates to a methylation gene marker for early detection of endometrial cancer, a multiplex PCR detection kit and application thereof. The methylation gene marker for early detection of endometrial cancer is selected from any at least one section of methylation region in any at least one gene of target gene ADARB2、ADCYAP1、ADHFE1、C17orf64、CDO1、GAD2、GRM7、GYPC、KCNA1、KCNQ5、NETO1、RYR2、SORCS3、STX16、TCTEX1D1、TMEM101 and ZSSCAN 12, and diagnosis of endometrial cancer is carried out by detecting the corresponding methylation region, so that the methylation gene marker has higher sensitivity and specificity, and has very important significance for improving the diagnosis rate of endometrial cancer high-risk groups, realizing early intervention treatment and reducing the death rate.

Description

Methylation gene marker combination for early detection of endometrial cancer, multiplex PCR detection kit and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a methylation gene marker for early detection of endometrial cancer, a multiplex PCR detection kit and application thereof.
Background
Endometrial cancer is an epithelial malignancy occurring in the endometrium, also known as endometrial cancer, accounting for about 20-30% of gynaecological malignancies. In recent years, the incidence rate of the tumor is gradually increased and has obvious younger trend, and the incidence rate of the tumor in the partially developed area reaches the first position of gynecological malignant tumor.
Since endometrial cancer has early symptoms of irregular vaginal bleeding and vaginal discharge, about 70% of endometrial cancer patients are diagnosed with early lesions confined to the uterine body. However, due to the non-specificity of the symptoms, there is still a large portion of patients who find them without the opportunity for early diagnosis. Early diagnosis is of great importance to endometrial cancer. On one hand, early diagnosis and early treatment can obviously improve the survival rate and the life quality of patients; on the other hand, with the trend of tumor rejuvenation, more endometrial cancer patients still have a need for fertility, and early findings will provide the possibility for conservation therapy.
There is currently no recommended means by which endometrial cancer can be routinely screened. According to the current diagnosis and treatment guidelines of endometrial cancer, vaginal ultrasound, serum tumor marker CA125 and the like are mainly used for auxiliary diagnosis clinically, but the method has the problem of low sensitivity and/or specificity, and is not suitable for the early conventional screening of endometrial cancer based on people. The endometrial cell collector can be used for screening endometrial lesions of symptom population and high-risk population by using liquid-based cytology examination, but is easily affected by operators, has limited sensitivity, and has certain traumata in sampling. For early screening of endometrial cancer, there remains a need to develop more accurate, more noninvasive markers.
DNA methylation is one of the epigenetic aspects, namely the addition of a methyl group at carbon number 5 of the cytosine base, and this modification is usually associated with silencing of the gene. DNA methylation is a key epigenetic regulator of gene expression, often resulting in defective gene expression. Increased methylation of tumor suppressor genes is an early event in many tumors. Methylation signature detection is currently a widely accepted source of tumor screening markers. In addition, DNA methylation detection is adopted as a tumor screening means, so that the method has the advantages of high sensitivity and specificity. But currently there is a lack of detection techniques, methods and products for endometrial cancer methylation gene detection. Therefore, it is highly desirable to provide a methylation-derived early screening marker which can distinguish endometrial cancer from non-cancerous groups, and the marker can be used for body fluid samples such as urine, blood, etc., sample collection of sloughed cells such as swabs, brush sheets, lavage fluid, etc., tumor tissue samples, etc., and background signal interference can be eliminated in various noninvasive sample collection to accurately distinguish endometrial cancer from detection targets of other diseases/healthy individuals.
Disclosure of Invention
In order to overcome the above-mentioned drawbacks and disadvantages of the prior art, a primary object of the present invention is to provide a methylation gene marker for early detection of endometrial cancer.
The invention discovers novel methylation genes ：ADARB2、ADCYAP1、ADHFE1、C17orf64、CDO1、GAD2、GRM7、GYPC、KCNA1、KCNQ5、NETO1、RYR2、SORCS3、STX16、TCTEX1D1、TMEM101 and ZSSCAN 12 which can be used for diagnosing endometrial cancer or precancerous lesions thereof, and the methylation of the genes is detected to diagnose endometrial cancer or precancerous lesions thereof, so that the sensitivity and the specificity are higher. The invention provides a new marker and a diagnosis strategy for the diagnosis of endometrial cancer and precancerous lesions thereof.
Another object of the invention is to provide a multiplex PCR detection kit for detecting early endometrial cancer screening diagnosis.
It is still another object of the present invention to provide the use of the above-mentioned methylation gene marker for preparing a diagnostic reagent for endometrial cancer.
It is still another object of the present invention to provide an application of the multiplex PCR detection kit as described above in the preparation of a diagnostic product for endometrial cancer.
The aim of the invention is achieved by the following scheme:
A methylation gene marker for early detection of endometrial cancer, selected from any one of at least one of the following methylation regions in any one of the target genes ADARB2、ADCYAP1、ADHFE1、C17orf64、CDO1、GAD2、GRM7、GYPC、KCNA1、KCNQ5、NETO1、RYR2、SORCS3、STX16、TCTEX1D1、TMEM101 and ZSCAN 12:
ADARB2 gene: chs 10: 1779052-1779183, chs 10: 1779756-1779910;
ADCYAP1 Gene: chr18:907522-907672, chr18:908206-908385;
ADHFE1 Gene: chr8: 67344466-67344599;
C17orf64 gene: chr17: 58498643-58498859, chr17: 58498879-58499081;
CDO1 gene: chr5: 115152259-115152414;
GAD2 gene: chr10: 26504914-26505063; chr10: 26506264-26506424; chr10: 26506907-26507068;
GRM7 gene: chr3: 6904068-6904274;
GYPC gene: chr2: 127413900-127414152;
KCNA1 gene: chr12: 5018675-5018893; chr12: 5019386-5019578;
KCNQ5 gene: chr6: 73331034-73331188; chr6: 73331252-73331411;
NETO1 Gene: chr18: 70535858-70535996;
RYR2 Gene: chr1: 237205894-237206034;
SORCS3 Gene: chs 10: 106400776-106400914, chs 10: 106401432-106401587;
STX16 Gene: chr20: 57225138-57225270;
TCTEX1D1 Gene: chr1: 67218010-67218198;
TMEM101 gene: chr17: 42092213-42092341;
ZSCAN12 gene: chr6: 28367209-28367379, chr6: 28367431-28367676.
According to the invention, by screening the related gene types of endometrial cancer and the methylation regions of each gene, seventeen marker genes (or target genes) and the corresponding functional optimal methylation regions are finally screened, the judgment limit value can be determined through the methylation results of the marker genes, the results of the methylation regions can be used for early detection of endometrial cancer, the accuracy of the results is very high, and auxiliary diagnosis references can be provided for clinicians.
The invention provides a novel marker which can be used as a diagnosis marker of endometrial cancer or precancerous lesions thereof, namely the methylation gene. Specifically, the invention uses at least one of the target gene ADARB2、ADCYAP1、ADHFE1、C17orf64、CDO1、GAD2、GRM7、GYPC、KCNA1、KCNQ5、NETO1、RYR2、SORCS3、STX16、TCTEX1D1、TMEM101 and ZCAN 12 as a target spot, diagnoses endometrial cancer by detecting the corresponding methylation region, has higher sensitivity and specificity, and has potential and prospect of being used as a marker for diagnosing or screening endometrial cancer. Therefore, at least one of the genes can be used as a biomarker of endometrial cancer, and has very important significance for improving the diagnosis rate of endometrial cancer high-risk groups, realizing early intervention treatment and reducing the death rate.
Furthermore, endometrial cancer is diagnosed by in vitro detection of a methylation region corresponding to any at least one target gene in the sample. The sample can comprise body fluid samples such as urine and blood, sample collected by sloughed cells such as swabs, brush pieces, lavage liquid, tumor tissue samples and the like.
Furthermore, when the sample is a sample collected by using sloughed cells such as a swab, a brush, a lavage liquid and the like, at least one target gene is used as a target point, and the endometrial cancer is diagnosed by detecting the corresponding methylation region, the sensitivity and the specificity can be both up to 100%.
Furthermore, when the sample is urine, a single target gene is used as a target point, and diagnosis is performed by detecting the corresponding methylation region, the sensitivity of up to 91% and the specificity of up to 95% can be still realized. The invention can be used for detecting urine samples by only one molecular marker (namely one gene), can achieve higher sensitivity and specificity, and provides a simpler, more convenient and more accurate method for detecting endometrial cancer.
Furthermore, diagnosis of endometrial cancer or precancerous lesions in the form of a combination of two or more of the above-described gene methylation can significantly improve sensitivity and specificity.
The invention also provides a detection primer for early detection of endometrial cancer, which is used for correspondingly detecting the methylation state of the methylation region of the marker gene, and the nucleotide sequence of the detection primer is as follows:
ADARB2 gene:
the corresponding detection primer of chr10: 1779052-1779183 is SEQ ID NO. 1-2;
the corresponding detection primer of chr10: 1779756-1779910 is SEQ ID NO 3-4;
ADCYAP1 Gene:
The corresponding detection primer of chr18:907522-907672 is SEQ ID NO. 5-6;
the corresponding detection primer of chr18:908206-908385 is SEQ ID NO 7-8;
ADHFE1 Gene:
The corresponding detection primer of chr8: 67344466-67344599 is SEQ ID NO 9-10;
C17orf64 gene:
the corresponding detection primer of chr17: 58498643-58498859 is SEQ ID NO. 11-12;
The corresponding detection primer of chr17: 58498879-58499081 is SEQ ID NO. 13-14;
CDO1 gene:
The corresponding detection primer of chr5: 115152259-115152414 is SEQ ID NO. 15-16;
GAD2 gene:
the corresponding detection primer of chr10: 26504914-26505063 is SEQ ID NO. 17-18;
the corresponding detection primer of chr10: 26506264-26506424 is SEQ ID NO. 19-20;
The corresponding detection primer of chr10: 26506907-26507068 is SEQ ID NO. 21-22;
GRM7 gene:
the corresponding detection primer of chr3: 6904068-6904274 is SEQ ID NO. 23-24;
GYPC gene:
The corresponding detection primer of chr2: 127413900-127414152 is SEQ ID NO. 25-26;
KCNA1 gene:
The corresponding detection primer of chr12: 5018675-5018893 is SEQ ID NO 27-28;
the corresponding detection primer of chr12: 5019386-5019578 is SEQ ID NO. 29-30;
KCNQ5 gene:
The corresponding detection primer of chr6: 73331034-73331188 is SEQ ID NO. 31-32;
The corresponding detection primer of chr6: 73331252-73331411 is SEQ ID NO. 33-34;
NETO1 Gene:
The corresponding detection primer of chr18: 70535858-70535996 is SEQ ID NO. 35-36;
RYR2 Gene:
the corresponding detection primer of chr1: 237205894-237206034 is SEQ ID NO. 37-38;
SORCS3 Gene:
The corresponding detection primer of chr10: 106400776-106400914 is SEQ ID NO 39-40;
the corresponding detection primer of chr10: 106401432-106401587 is SEQ ID NO. 41-42;
STX16 Gene:
The corresponding detection primer of chr20: 57225138-57225270 is SEQ ID NO. 43-44;
TCTEX1D1 Gene:
The corresponding detection primer of chr1: 67218010-67218198 is SEQ ID NO. 45-46;
TMEM101 gene:
The corresponding detection primer of chr17: 42092213-42092341 is SEQ ID NO. 47-48;
ZSCAN12 gene:
the corresponding detection primer of chr6: 28367209-28367379 is SEQ ID NO. 49-50;
the corresponding detection primer of chr6: 28367431-28367676 is SEQ ID NO. 51-52.
The invention also provides a multiplex PCR detection kit for detecting early endometrial cancer screening diagnosis, which comprises reagents for detecting target gene methylation;
the target gene is selected from at least one ：ADARB2、ADCYAP1、ADHFE1、C17orf64、CDO1、GAD2、GRM7、GYPC、KCNA1、KCNQ5、NETO1、RYR2、SORCS3、STX16、TCTEX1D1、TMEM101 of the following genes and ZSSCAN 12.
The reagent includes at least one of the above-described detection primers.
Further, the working process of the multiplex PCR detection kit may be:
(1) Extracting DNA in a sample to be detected, and converting to obtain modified DNA;
(2) Detecting the modified DNA by adopting a multiplex PCR detection kit, wherein the detection procedure comprises two rounds of PCR reactions; the primer of the first round of multiplex PCR comprises a target region amplification primer segment, and a sequencing connecting primer segment of a forward sequencing primer and a reverse sequencing primer; the primers of the second round of multiplex PCR include a complementary segment comprising a segment that is ligated to the sequencing primer in the first round of PCR primers and another segment comprising a sequencing adapter.
In the working process, the transformation means that DNA methyl transformation is realized; the reagents used are those conventionally used for methyl conversion, and may include, but are not limited to, bisulfite or hydrazine salt treatment, enzymatic conversion, and the like.
Further, the amplified product obtained by the two PCR reactions was subjected to second generation sequencing.
Further, the length of the amplified primer fragment of the targeting region is 26-38bp.
Further, the forward detection primer obtained by the first round of multiplex PCR comprises a forward sequencing primer + NNNNNNNN + forward amplification primer, and the length of the forward detection primer can be 63-75bp; the reverse detection primer comprises a reverse sequencing primer and a NNNNNNNN + reverse amplification primer, and the length of the reverse detection primer can be 69-72bp; and can also be adjusted according to the tester.
Second generation sequencing can be performed directly using an Illumina sequencer (sequencing modes such as PE150, PE250, SE150, SE250, etc. can be used).
The invention also provides application of the methylation gene marker in preparation of endometrial cancer diagnosis products.
The invention also provides application of the multiplex PCR detection kit in preparation of endometrial cancer diagnosis products.
The diagnostic product includes any one of a kit, a formulation, and a chip.
Compared with the prior art, the invention has the following advantages:
The invention can remarkably improve the sensitivity and specificity of early endometrial cancer detection by screening the related gene types of endometrial cancer and the methylation region of each gene and finally screening the seventeen marker genes (or called target genes) and the corresponding functionally optimal methylation regions. The methylation gene marker can be used for body samples such as urine and blood, sample collection of sloughed cells such as swabs, brush sheets and lavages, tumor tissue samples and the like, and can eliminate background signal interference in various samples to accurately distinguish detection targets of endometrial cancer and other diseases/healthy individuals. For a urine sample collected in a noninvasive way, even if the concentration of the DNA template is low, the methylation gene marker can realize excellent detection sensitivity and specificity, reduce unnecessary wound sampling, have the advantages of noninvasive sampling, higher sensitivity and specificity and the like, not only optimize the distribution of medical resources, improve the acceptance of a subject and improve screening experience, but also ensure the accuracy of results so as to help realize the purposes of early discovery, early diagnosis and early treatment of endometrial cancer.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a kit detection flow diagram for multiplex PCR amplified methylation sequencing for sequencing multiple methylation regions.
FIG. 2 is a flow chart of library construction for multiplex PCR amplified methylation sequencing.
FIG. 3 is a graph showing the effect of DNA fragmentation on the methylation sequencing effect of multiple targeted amplicons.
FIG. 4 is a graph showing the effect of sample starting DNA loading on the methylation sequencing effect of multiple targeted amplicons.
FIG. 5 shows the results of the resemblance of multiplex targeted amplicon methylation sequencing with DNA methylation chip technology.
FIG. 6 is a graph showing methylation rate levels of gene methylation markers in endometrial cancer tissue, paracancerous tissue, and non-cancerous normal tissue.
FIG. 7 is a graph showing changes in methylation rates of gene methylation markers in pre-and post-endometrial cancer paired samples.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. The materials referred to in the examples below are available commercially unless otherwise specified. The method is conventional unless otherwise specified.
Example 1: screening of methylation Gene markers for endometrial cancer
The invention uses TCGA public database to obtain 450K methylation chip data of 524 cancer tissues and 34 cancer tissues beside endometrial cancer, and screens the sites of obvious hypermethylation in endometrial cancer tissues; screening the differential sites by adopting a CHAMP.DMP method, and screening 8730 differential sites by taking the P value after FDR correction of <0.05 and the average methylation rate of cancer tissues and the average methylation rate of paracancerous tissues of >0.35 as screening criteria.
Since urine, cervical brush, vaginal swab, blood and other sampling methods collect cells or DNA derived from tumor, the incorporation of white blood cells or urothelial cells and other components is inevitable. The incorporation of these non-target exogenous DNA may interfere with the detection of the target marker as a background signal. Therefore, the interference of the background signal should be carefully handled in screening markers. In view of the above, the present invention further incorporates 78 cases of paraurinary tract cancer tissue and 338 cases of leucocyte 450k methylation chip data derived from females in TCGA and GEO databases, and performs champ. Dmp difference test with 425 cases of endometrial cancer tissue data, respectively. Sites meeting the following conditions were included in the screening range at the same time: (1) two difference tests with FDR corrected P value <0.05 and; (2) Cancer tissue average methylation rate-paraurinary tract cancer tissue average methylation rate >0 and; (3) Cancer tissue average methylation rate-leucocyte paracancestral tissue average methylation rate >0 and; (4) average methylation rate beside urinary tract cancer <0.1 and; (5) the average methylation rate of leukocytes is <0.1. The sites meeting the above requirements were intersected by the differential sites of endometrial cancer/paracancerous differential screening, and a total of 1464 sites were included in the next screening process.
In order to further evaluate the methylation level of the 1464 candidate sites in the actual sample, the invention carries out Illumina Epic 850k chip detection on 12 endometrial cancer patients and 15 control exfoliated cell samples collected in university tumor prevention centers in Zhongshan and first people hospitals in Fushan city. The chip is an upgrade version of 450K chip, and almost 40 ten thousand methylation sites are newly added while most 450K chip sites are reserved. The selection of 17 genes from C17orf64、CDO1、KCNQ5、STX16、ZSCAN12、KCNA1、ADARB2、SORCS3、GAD2、RYR2、GRM7、ADCYAP1、NETO1、GYPC、TMEM101、ADHFE1 and TCTEX1D1 was finally determined. Methylation sites and effect information of the genes with optimal endometrial cancer diagnosis effect are shown in table 1.
The area under the curve of the optimal site for 17 genes is between 0.867 and 0.983. Wherein the AUC of 7 genes including C17orf64, CDO1, KCNQ5, STX16, ZSCAN12, KCNA1 and ADARB is greater than 0.98; the AUC of 7 genes including SORCS, GAD2, RYR2, GRM7, ADCYAP1, NETO and GYPC is between 0.90 and 0.95. Different genes show different advantages in detection sensitivity and specificity, wherein the sensitivity of CDO1, KCNA1 and TCTEX D1 genes can reach 100%, and the specificity of C17orf64, KCNQ5, STX16 and ZCAN 12 genes can reach 100%. The sensitivity of the 17 gene methylation markers except GRM7 and GAD2 is higher than 80%; the specificity was higher than 80% except TCTEX D1. Taken together, the sensitivity of the C17orf64, KCNQ5, STX16 and ZSCAN12 genes can reach 91.7% when the specificity is 100%; the specificity of KCNA1 gene can reach 93.3% when the sensitivity is 100%, and the gene has excellent discrimination capability between endometrial cancer and contrast.
In addition, sites with strong discrimination are clustered around the optimal site, and within 500bp upstream and downstream of the optimal site, the AUC of the gene with 1-8 sites is more than 0.8, wherein the AUC of the 14 genes with at least 3 sites and more is more than 0.8. Genes with AUC >0.8 with fewer than 3 sites are also most likely due to the small probe coverage density of these genes and the few detection sites in the design of the Epic 850k chip. The occurrence of multi-site clustering further indicates the certainty of the gene methylation as an endometrial cancer diagnosis marker, and is beneficial to the development of a methylation site detection method. The results show that 17 genes of the invention have excellent identification ability in endometrial cancer and control populations.
TABLE 1
Remarks: a is the number of sites with AUC >0.8 within 500bp upstream and downstream of the optimal site.
Example 2: construction of multiplex PCR amplified methylation sequencing technique to detect marker methylation Signal
For 17 genes screened in the example 1, a DNA amplicon targeted methylation sequencing method is established, target PCR amplification is carried out on the DNA converted by the re-sulfite, and the methylation level of the candidate marker in the sample is obtained by detecting through second generation sequencing, and the flow chart is shown in figure 1. The method comprises the following specific steps:
2.1 Design of target region multiplex PCR primer
Determination of the target area: the sites on CpG islands often appear consecutively, and in order to determine the optimal sequence amplification range, the invention firstly performs optimal amplification region selection on the candidate 17 differential methylation sites by taking genes as units. Taking the optimal site of the area under the curve (AUC) of the working characteristic curve of the subject in the gene as the center, extracting the methylation rate of all 850k chips on the gene containing methylation sites in the data of the exfoliated cells and endometrial cancer tissues, and pulling out the region with the denser 150-350bp methylation sites and the most obvious difference in the exfoliated cells and tissues for the next primer design.
Design of target region PCR primer: primer design of each sequence was performed using DNA methylation specific PCR primer design software such as METHPRIMER. And pairing the designed primers in pairs, and comparing compatibility of paired primers to avoid the problems of primer dimers, hairpin structures and the like among a plurality of pairs of primers as much as possible, thereby improving the success rate of the primers. Through repeated debugging, 26 pairs of primers are designed together for 17 target genes to carry out multiplex PCR detection. The primer sequences, the chromosomal physical location of the amplified region and the information on the target gene are shown in Table 2. Wherein "F" represents a forward amplification primer and "R" represents a reverse amplification primer.
TABLE 2
2.2 Two rounds of PCR amplification to construct an amplicon library of the target fragment and sequencing the amplicon library on a machine
Pretreatment of urine samples and DNA extraction were performed using ZYMO Quick-DNA ™ Urine Kit, and DNA bisulfite conversion was performed using ZYMO EZ DNA Methylation-Gold Kit. For DNA samples after bisulfite conversion, the invention establishes a multiplex PCR primer system for 26 fragments of 17 target genes, and 280 methylation sites can be detected cumulatively. The method is divided into two rounds of PCR: the primer of the first round of multiplex PCR is divided into two parts, namely a target region amplification primer fragment of 26-38 bp, a sequencing connecting primer fragment of 29bp (forward primer) and 26bp (reverse primer); the length of the obtained first round multiplex forward PCR primer is 63-75bp, and the length of the reverse PCR primer is 69-72bp. The primer of the second round of multiplex PCR comprises a complementary segment to the ligation segment of the sequencing primer in the primer of the first round of PCR and another segment of the primer comprising a sequencing adapter. The amplified products after two rounds of PCR can be directly subjected to second generation sequencing by using an Illumina sequencer (the sequencing modes such as PE150, PE250, SE150, SE250 and the like can be adopted). The design principle of the two rounds of PCR primers is shown in FIG. 2.
The method comprises the following specific steps:
(1) First round PCR amplification
The first round of PCR is a multiplex PCR amplification system, and the amplification primers are divided into two parts: { sequencing primer fragment+8 free bases+amplification primer fragment }. The kit covers 28 methylation areas including 17 genes, and the sequence of amplified primer fragments of each amplification area is shown in Table 2. The basic information of each methylation detection region, the reference genome position of the detection sequence, the length of the detection sequence and the reference genome sequence of the detection region are shown in Table 3.
TABLE 3 Table 3
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The primer sequence design concept of the first round multiplex PCR primer is shown in Table 4, and the primer overall composition is "sequencing primer+ NNNNNNNN +amplification primer". Forward sequencing primer sequence 5'-AATGATACGGCGACCACCGAGATCTACAC-3'; adding eight variable bases NNNNNNNN in the middle to serve as a barcode sequence for distinguishing sequence sample information obtained by sequencing; the methylation forward amplification primers of each gene were then ligated and the F primers shown in Table 2 were described. The reverse sequencing primer sequence is 5'-CAAGCAGAAGACGGCATACGAGATTC-3'; eight variable bases 'NNNNNNNN' are added in the middle to be used as barcode for distinguishing sequence sample information obtained by sequencing; the methylation reverse amplification primers of each gene were then ligated and the R primers shown in Table 2 were described. The invention mixes the first round multiplex PCR primer of each amplified region according to equal proportion to be used as a PCR primer, uses DNA converted by bisulfite as a template DNA and uses KAPA HiFi Mix as DNA polymerase to amplify. The number of amplification cycles is controlled within 15-35 cycles according to the mass and concentration of the DNA template. Wherein, the PCR reaction system comprises the following components (the dosage per hole): 2. mu L5 x KAPA HiFi buffer;0.3 Mu L10 mM dNTPs;0.5 2, L DMSO;0.2 A [ mu ] L KAPA HIFI Polymerase; 1.2, a [ mu ] L forward sequencing primer (5 mu M); 1.2, a [ mu ] L reaction sequencing primer (5 [ mu ] M); 3.2, a [ mu ] L enzyme-free water; 2. and [ mu ] L transformed DNA. PCR reaction conditions: firstly, 95-5 min; then 98-20 s, 50-60-30 s, 72-1 min,15-35 cycles; finally, the temperature is maintained at 4 ℃.
TABLE 4 Table 4
(2) Second round PCR amplification and purification
Purifying the amplified product of the first round of PCR by using AMPure XP magnetic beads, taking the purified nucleic acid as a template DNA of the second round of PCR, carrying out the second round of amplification, using KAPA HiFi Mix as DNA polymerase for amplification, and controlling the amplification annealing temperature and the extension time within 10-20 cycles according to the specific conditions of the product. The forward primer sequence for the two rounds of amplification was CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG and the reverse primer sequence was AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC, where "NNNNNNNN" is the sequencer index sequence. It should be noted that the first round of PCR sequencing primer and the second round of sequencing primer can be modified according to specific requirements of different brands and different specifications of the sequencer, and the sequencing primer provided in the invention only provides an example of one of the sequencing primers; the use of other sequencing primers that are appropriate for the corresponding sequencer does not affect the detection results of the present kit. Wherein, the PCR reaction system comprises the following components (the dosage per hole): 2. mu L5 x KAPA HiFi buffer;0.3 Mu L10 mM dNTPs;0.5 2, L DMSO;0.2 A [ mu ] L KAPA HIFI Polymerase; 1. 2, a [ mu ] L forward sequencing primer (5 mu M); 1. 2, a [ mu ] L reaction sequencing primer (5 [ mu ] M); 3. 2, a [ mu ] L enzyme-free water; 2. and [ mu ] L of the first PCR dilution product DNA. PCR reaction conditions: firstly, 95-5 min; then 98-20 s, 50-60-30 s, 72-1 min,10-20 cycles; finally, the temperature is maintained at 4 ℃.
(3) Mixing and quality evaluation of amplified products
And (3) purifying the amplified products of the second round of PCR by using AMPure XP magnetic beads, quantifying nucleic acid by using a qubit DSDNA HIGH SENSITIVITY ASSAY KIT and a qPCR method on the DNA obtained by purification, mixing the amplified products of different samples with equal molar mass, and performing second generation sequencing on a corresponding sequencer.
(4) Bioinformatics analysis of sequencing data
After the sequencing data is taken off the machine, carrying out data quality check by using fastqc software, and removing the sequencing joint and low-quality base and sequence by using software Trimmomatic to obtain a qualified sequence after quality control. Subsequently, the qualified sequence and the target sequence of the amplified region were aligned using basmap methylation sequencing data alignment software. Then, the methylation level of the CG base on each target sequence of each sample is calculated, and the calculation formula of the methylation level is: methylation level of C site = number of sequences supporting methylation/(number of sequences supporting methylation + number of sequences supporting non-methylation); the methylation rate levels of all methylation sites in the detection region for each sample can be obtained according to the above formula. For sequences with a sequencing depth of less than 50X at a site, the methylation rate at that site of the sample is defined as the deletion value NA and is no longer included in the calculation.
(5) Evaluation of methylation sequencing Effect of target amplicon
① Effect of DNA fragmentation on multiplex PCR amplification
Since DNA from samples such as exfoliated cells is always subject to the risk of degradation and fragmentation of nucleic acid, 5 samples with different degrees of fragmentation are tested, and the influence of DNA fragmentation on the detection result of the kit is observed. The results show that for samples with severe fragmentation, the percent of qualified sites detected by the targeted methylation sequencing technology of the invention is more than 99%, which indicates that the detection method of the invention is not basically affected by DNA fragmentation, and reliable detection data can be obtained under the condition of severe fragmentation, as shown in fig. 3 and table 5.
TABLE 5
② Effect of loading on multiplex PCR amplification sequencing
The sample of noninvasive collection such as the exfoliated cells is large in individual sampling heterogeneity, and the situation that the obtained sample is insufficient in nucleic acid amount often exists, so that the detection effect under the condition of different sample initial DNA loading amounts is tested. The results showed that even with DNA input less than 200ng, the average percent of qualifying sites detected by targeted methylation sequencing techniques could still reach 98% and there was no statistical difference between the percent of qualifying sites grouped with higher sample starting DNA loading (fig. 4). This demonstrates that the detection method of the present invention can still obtain sufficient data in the case where only a small amount of nucleic acid is obtained from the sample to be tested.
③ Resemblance of multiplex targeted amplicon methylation sequencing to DNA methylation chip technology
Different detection methods can have certain deviation on the detection results of methylation sites. In order to evaluate the effect of the multiplex targeted amplicon methylation sequencing constructed by the application on the reflection of the real methylation level in the samples, 3 samples subjected to multiplex targeted amplicon methylation sequencing are detected by adopting an Illumina Epic 850K methylation chip. Epic 850K methylation chips are one of the widely used and accepted DNA methylation detection methods. The correlation of methylation rates of the two detection methods for these 3 retest samples was calculated by comparing the methylation rates of the methylation sites detected by both methods. The results show that the two detection methods of the 3 samples have good retest consistency, and the correlation coefficient R 2 is 0.878,0.941 and 0.851 respectively, and have very high positive correlation (figure 5). This illustrates that the detection method of the present application can reflect the true methylation signal in the sample.
In conclusion, the multiplex targeting amplicon methylation sequencing detection method disclosed by the invention can be compatible with common problems of serious DNA fragmentation, low DNA input and the like, can reflect the real methylation rate level in the sample, has the feasibility of developing a detection kit, and has the advantages especially when detecting samples with larger individual heterogeneity such as exfoliated cells, body fluid source samples and the like.
Example 3: diagnostic effect of methylation markers on endometrial cancer in urine samples
Urine samples are completely non-invasive compared to blood samples and tissue samples; compared with the cervical brush sheet, vaginal swab and other samples, the method is less influenced by individual sampling techniques, and the method is high in acceptance of the sampling mode by the masses without assistance of medical staff, so that home sampling can be realized. The test samples in this example were urine from 107 endometrial cancer patients and 111 control participants collected at the university of chinese university tumor prevention center, the university of guangzhou medical science affiliated tumor hospital.
The sample is subjected to DNA extraction, DNA bisulfite modification, targeted methylation amplification library construction and sequencing by an Illumina platform by adopting the detection method constructed in the embodiment 2 of the application. Sequencing data is subjected to the biological information analysis flow to obtain the methylation levels of the methylation sites in the 17 gene amplification regions. 280 site information from 17 genes was included in the analysis via the data quality control procedure. Because the data is subjected to quality control filtration according to the sequencing depth of each gene, the sites with the sequencing depth less than 50 sequences are defined as deletion, the number of cases which are finally included into analysis at each site after the filtration is different from 97 to 106, and the number of comparison cases is different from 105 to 111.
Methylation loci and effect information of methylation loci with optimal endometrial cancer diagnosis effect in independent samples by using a targeted amplification methylation sequencing mode are shown in Table 6. Methylation sites detected in 17 genes varied from 3 to 35. When the corresponding gene was measured, all primer pairs covered by the gene in Table 2 were used for detection. By modeling the methylation site in the same gene by using a Logistic model, the area under the curve is between 0.788 and 0.886. Wherein the AUC of the 3 gene methylation markers including GAD2, ADCYAP1 and C17orf64 is greater than 0.85; AUC of 16 markers other than CDO1 was greater than 0.8. Similar to the results in example 1, CDO1 exhibited excellent sensitivity of 91.3%; the sensitivity of GAD2 and GRM7 is higher than 80%; the specificity of KCNA1 and ADCYAP1 is higher than 90%. The area under the curve of the methylation site with the optimal prediction effect in the 17 gene methylation detection regions is 0.730-0.814.
TABLE 6
Remarks: a is the sample size of case group inclusion modeling; b is the amount of the modeling sample taken in by the control group; AUC 1 is the area under the modeling curve of all loci of the gene; c is the number of methylation sites identified in the amplified region; AUC 2 is the area under the curve predicting the site of optimal methylation.
Of the 17 gene methylation markers of the present invention, GAD2, ADCYAP1, C17orf64, etc. have the best overall performance, GAD2, etc. have the advantage in sensitivity, KCNA1, ADCYAP1, etc. have the advantage in specificity, and thus the diagnostic efficacy of the combination marker against 107 cases of endometrial cancer and 111 cases of controls was further evaluated. When performing the polygenic marker joint detection, all primer pairs covered by the joint gene in table 2 were used for detection. Table 7 shows the discrimination effect when two, three and four gene markers are combined. Specifically, when two gene methylation markers are combined, the AUC of the combination of the GAD2 marker and the GYPC marker is the highest, the sensitivity is up to 0.928, the specificity is 86.8%, and the effect is obviously improved compared with that of a single gene methylation marker. The number of the other genes can reach 0.9 when the genes are combined two by two, and the excellent combined diagnosis performance is shown. When three gene methylation markers are combined, the combined AUC of the three markers of C17orf64, GAD2 and GYPC is highest, the sensitivity is up to 0.948, the sensitivity is 85.4%, the specificity is 91.2%, the sensitivity of the model is obviously improved compared with that of a model with two markers, and the accuracy is improved. Wherein, the AUC of the combination of GAD2 and GYPC with C17orf64, ADCYAP1, TMEM101, ZCAN 12 and other genes can reach more than 0.94, and the AUC of the combination of ADARB2, CDO1, GRM7, KCNA1, NETO1, SORCS3, STX16 and TCTEX1D1 genes with C17orf64, GAD2 or GYPC can also reach 0.93. The diagnostic performance at the four methylation gene marker combinations can be further improved, wherein the AUC of the marker combinations of ADCYAP, GAD2, GYPC and TMEM101 can reach 0.959, the sensitivity is 93.9%, and the specificity is 86.1%. In a better four-marker combination model, the highest occurrence frequency of genes GAD2, GYPC and TMEM101 is the key gene in the combination model, and the mutually combined AUC of genes such as GAD2, GYPC, ADCYAP1, TMEM101, C17orf64, ADHFE1, ZCAN 12, TCTEX1D1, STX16, RYR2, KCNQ5, SORCS3, RYR2 and KCNA1 is close to 0.95, so that the gene complementarity is shown.
TABLE 7
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When five methylation gene markers were included, the AUC of all combinations was higher than 0.857, the optimal combination was GAD2, GYPC, ADCYAP1, TMEM101 and STX16, AUC was 0.960; AUC of GAD2, GYPC, ADCYAP1, C17orf64, ZSCAN12 gene combination was also 0.960; 130 combinations have AUC higher than 0.95, and 17 gene markers in 130 combinations are all incorporated into the model for multiple times, so that the importance of each gene in a high-precision model is fully reflected.
When the markers are combined by six genes, the AUC of all the combinations is higher than 0.872, the optimal combinations are ADCYAP1, C17orf64, GAD2, GYPC, TCTEX1D1 and ZSSCAN 12, and the AUC is 0.962; wherein the AUC of all combinations with the pre-effect of 100 is higher than 0.956, 17 gene markers are incorporated into the model for multiple times, and the importance of each gene in a high-precision model is fully reflected.
When seven genes are combined into the marker, the AUC of all the combinations is higher than 0.884, and the optimal combinations are ADCYAP1, C17orf64, GAD2, GYPC, TCTEX1D1, TMEM101 and ZCAN 12, and the AUC is 0.963; the same diagnostic effect can be achieved by the combination ADARB, ADCYAP1, GAD2, GYPC, STX16, TCTEX1D1 and TMEM 101; in the seven gene combinations, the AUC of all combinations with the effect of 100 is higher than 0.960, 17 gene markers are incorporated into the model for many times, and the importance of each gene in a high-precision model is fully reflected.
When the markers are combined by eight genes, the AUC of all the combinations is higher than 0.888, and the optimal combinations are ADCYAP1, C17orf64, GAD2, GYPC, RYR2, TCTEX D1, TMEM101 and ZCAN 12, and the AUC is 0.964; in the eight-gene combination, the AUC of all combinations with the effect of 100 is higher than 0.961, 17 gene markers are repeatedly included in the model, and the importance of each gene in a high-precision model is fully reflected.
In conclusion, the methylation gene marker or the combination thereof can well distinguish endometrial cancer from control individuals, and can be used for preparing a kit for diagnosing endometrial cancer. And the endometrial cancer or precancerous lesions are diagnosed in the form of the combination of two or more of the above genes methylation, so that the sensitivity and the specificity can be remarkably improved.
Notably, of the 100 cases of endometrial cancer with definite pathological stage in the case group, as many as 73% of patients in stage I (extremely early stage) and 19% of patients in stage II and stage IV (middle and late stage) represent only 8%, which shows excellent performance of the above markers on early endometrial cancer, and can be used for early diagnosis of endometrial cancer.
Example 4: diagnostic effect of methylation markers on endometrial cancer in cervical brush sample
To evaluate the diagnostic value of 17 markers contained in example 1 for endometrial cancer in a sample of cervical brush material, the example test samples were 17 endometrial cancer patients and 14 control participants collected at the university center for tumor control and the first-people hospital in berg. The cervical brush sheet is a relatively noninvasive sampling scheme, can perform self-sampling, and has the advantage of being used as a noninvasive diagnostic sampling sample. The sample is subjected to DNA extraction, DNA bisulfite modification, targeted methylation amplification library construction and sequencing by an Illumina platform by adopting the detection method constructed in the embodiment 2 of the application. Sequencing data is subjected to the biological information analysis flow to obtain the methylation levels of the methylation sites in the 17 gene amplification regions. 280 site information from 17 genes was included in the analysis via the data quality control procedure. Because the data is subjected to quality control filtration according to the sequencing depth of each gene, the sites with the sequencing depth less than 50 sequences are defined as deletion, the number of cases which are finally included into analysis at each site after the filtration is 14-17, and the number of comparison is 13-14.
The diagnostic effect of the genes contained in the application on endometrial cancer in cervical brush sample is shown in Table 8. The methylation sites detected in the 17 genes were between 3 and 35. When the corresponding gene was measured, all primer pairs covered by the gene in Table 2 were used for detection. And (3) carrying out Logistic model modeling on methylation sites in the same gene, and taking an optimal cut-off value to evaluate the discrimination accuracy of each gene on individuals in a case group and a control group. The 11 genes methylation markers can completely and accurately judge endometrial cancer and control individuals, and the genes comprise ADARB, ADCYAP1, C17orf64, GAD2, GYPC, KCNA1, KCNQ5, SORCS3, STX16, TMEM101 and ZCAN 12. In addition, the GRM7, TCTEX D1, CDO1 and NETO are completely correct for the comparison group, and the RYR2 is correct for the case group, but the specificity of the two methylation markers can reach 100%, so that the method has good effect in the recognition of the comparison. The above results indicate that 17 gene methylation markers of the present application have excellent diagnostic ability for endometrial cancer when a cervical brush sample is used as a test sample.
TABLE 8
Remarks: item A is the case group inclusion modeling sample size; b is the amount of the control group inclusion modeling sample; c, judging the case group correctly, and obtaining a sample/total sample; d is the correct judgment of the control group, and the sample/total sample; e is the number of methylation sites identified by the amplified region; AUC 1 is the area under the modeling curve of all loci of the gene; AUC 2 is the area under the curve predicting the site of optimal methylation.
Example 5: diagnostic effect of methylation markers on endometrial cancer in vaginal swab samples
To evaluate the diagnostic value of 17 markers contained in example 1 for endometrial cancer in cervical brush sample, the test samples of this example were vaginal swabs of 17 endometrial cancer patients and 14 control participants collected at the university center for tumor control and the first-people hospital in berg. Compared with a cervical brush piece, the vaginal swab has shallower sampling depth, and smaller assistance requirements of medical staff, and is one of non-invasive, simple and easy-to-operate sampling modes. The sample is subjected to DNA extraction, DNA bisulfite modification, targeted methylation amplification library construction and sequencing by an Illumina platform by adopting the detection method constructed in the embodiment 2 of the application. Sequencing data is subjected to the biological information analysis flow to obtain the methylation levels of the methylation sites in the 17 gene amplification regions. 280 site information from 17 genes was included in the analysis via the data quality control procedure. Because the data is subjected to quality control filtration according to the sequencing depth of each gene, the sites with the sequencing depth less than 50 sequences are defined as deletion, the number of cases which are finally included into analysis at each site after the filtration is 15-17, and the number of comparison is 11-14.
The diagnostic effect of the genes contained in the application on endometrial cancer in a vaginal swab sample is shown in Table 9. The methylation sites detected in the 17 genes were between 3 and 35. When the corresponding gene was measured, all primer pairs covered by the gene in Table 2 were used for detection. By modeling the methylation site in the same gene by using a Logistic model, the area under the curve is 0.874-1. The 13 gene methylation markers can completely and accurately judge endometrial cancer and control individuals, the area under the curve is 1, and the sensitivity and the specificity are 100%. These gene methylation markers include ADARB, ADCYAP1, C17orf64, GAD2, GYPC, KCNA1, KCNQ5, RYR2, SORCS3, STX16, TCTEX1D1, TMEM101 and ZCAN 12. In addition, AUC of GRM7 and CDO1 genes was greater than 0.95, showing excellent diagnostic efficacy. The AUC of NETO and ADHFE are respectively 0.929 and 0.874, the specificity of the two gene methylation markers can reach 100%, and the gene methylation markers have good capability in recognition of comparison. The above results indicate that 17 gene methylation markers of the present application have excellent diagnostic ability for endometrial cancer when a vaginal swab sample is used as a test sample.
TABLE 9
Remarks: item A is the case group inclusion modeling sample size; b is the amount of the control group inclusion modeling sample; c, judging the case group correctly, and obtaining a sample/total sample; d is the correct judgment of the control group, and the sample/total sample; e is the number of methylation sites identified by the amplified region; AUC 1 is the area under the modeling curve of all loci of the gene; AUC 2 is the area under the curve predicting the site of optimal methylation.
Example 6: detection effect of methylation marker in endometrial cancer tissue and paracancerous sample
To evaluate the signals of 17 markers contained in example 1 in endometrial cancer tissues, the test samples of this example were 88 endometrial cancer tissues derived from endometrial cancer patients, 34 paracancerous tissues derived from endometrial cancer patients, and 20 endometrial normal tissues derived from benign myoma patients collected at the university of Zhongshan tumor control center and the first-people hospital in berg city. Methylation signals in endometrial cancer tissue can reflect the reality of the primary foci, and provide reliable verification for the marker signals in the application. The sample is subjected to DNA extraction, DNA bisulfite modification, targeted methylation amplification library construction and sequencing by an Illumina platform by adopting the detection method constructed in the embodiment 2 of the application. Sequencing data is subjected to the biological information analysis flow to obtain the methylation levels of the methylation sites in the 17 gene amplification regions. 280 site information from 17 genes was included in the analysis via the data quality control procedure.
The application comprehensively compares methylation rate differences of 17 gene methylation markers in cancer and paracancerous tissues and cancer and normal tissues, and uses a sign rank sum test to conduct inter-group difference analysis. The results showed that the methylation rate of cancer tissue was significantly higher in all sites than in normal tissue among 280 methylation sites covered with 17 genes (P value range: 6.4X10 -11- 6.8×10-3, P value median: 5.4X10 -9); the methylation rate of the cancer tissue of 246 sites is obviously higher than that of the other sites, and the methylation rate of the cancer tissue is 84 percent. FIG. 6 shows the methylation rate distribution of representative methylation sites in 17 genes in normal, paracancerous and cancerous tissues. As shown in the figure, the methylation rate of each methylation site in cancer tissues is obviously higher than that of normal tissues and tissues beside the cancer, and the methylation rate is particularly obvious in genes such as GAD2, GRM7, SORCS3, TMEM101, KCNA1, GYPC, CDO1, C17orf64 and the like, and the P value of the cancer-to-cancer difference is less than 1X 10 -9.
Example 7: methylation marker trend comparison before and after surgery
If the markers of the application can accurately reflect the presence of endometrial cancer lesions, the methylation signature will theoretically be significantly reduced or even vanished after surgical removal of the lesions. To further increase the evidence of the authenticity of the markers on this level, the present example collected urine samples from 36 patients at the center for tumor control at the university of Zhongshan and the first-person hospital in berg for 3-5 days before and after surgery for dynamic monitoring of the markers in the present application. Since endometrial cancer surgery is usually performed by using double uterine attachments, it is not convenient to collect cervical brush sheets and vaginal swabs for postoperative patients, so that more noninvasive urine samples are used as the type of the monitored samples. Since the tumor-associated methylation signature released by the lesion after the tumor resection will terminate, the methylation signature of the patient after the operation should theoretically be reduced.
Methylation rate levels of 17 gene methylation markers in preoperative and postoperative paired samples of the same patient were comprehensively compared, and inter-group differential analysis was performed using a symbol rank and paired test. As a result, it was found that, of 280 methylation sites covered by the 17 gene methylation marker detection, the methylation rate of cancer tissue with 235 sites was significantly higher than that of the paracancer, and the methylation rate was 80%. FIG. 7 shows the methylation rate distribution of representative methylation sites in 17 genes in pre-and post-operative samples. As shown in the figure, the methylation rate of each methylation site in the post-operation sample is significantly reduced compared with the pre-operation sample, and representative sites of the remaining 15 genes except STX16 reach significant levels, and the P value is between 1.7X10 -3~1.7×10-5. Taken together, pre-operative/post-operative differences in the methylation sites ADHFE1, TMEM101, KCNA1, ADCYAP, and GYPC are particularly evident, with P values less than 1 x 10 -4 levels.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. A methylation gene marker for early detection of endometrial cancer, characterized by at least one methylation region selected from any one of the genes ADARB2、ADCYAP1、ADHFE1、C17orf64、CDO1、GAD2、GRM7、GYPC、KCNA1、KCNQ5、NETO1、RYR2、SORCS3、STX16、TCTEX1D1、TMEM101 and ZSCAN12 of interest:
ADARB2 gene: chs 10: 1779052-1779183, chs 10: 1779756-1779910;
ADCYAP1 Gene: chr18:907522-907672, chr18:908206-908385;
ADHFE1 Gene: chr8: 67344466-67344599;
C17orf64 gene: chr17: 58498643-58498859, chr17: 58498879-58499081;
CDO1 gene: chr5: 115152259-115152414;
GAD2 gene: chr10: 26504914-26505063; chr10: 26506264-26506424; chr10: 26506907-26507068;
GRM7 gene: chr3: 6904068-6904274;
GYPC gene: chr2: 127413900-127414152;
KCNA1 gene: chr12: 5018675-5018893; chr12: 5019386-5019578;
KCNQ5 gene: chr6: 73331034-73331188; chr6: 73331252-73331411;
NETO1 Gene: chr18: 70535858-70535996;
RYR2 Gene: chr1: 237205894-237206034;
SORCS3 Gene: chs 10: 106400776-106400914, chs 10: 106401432-106401587;
STX16 Gene: chr20: 57225138-57225270;
TCTEX1D1 Gene: chr1: 67218010-67218198;
TMEM101 gene: chr17: 42092213-42092341;
ZSCAN12 gene: chr6: 28367209-28367379, chr6: 28367431-28367676.
2. A detection primer for early detection of endometrial cancer, characterized in that: a nucleotide sequence for a corresponding detection of the methylation status of the methylation region of the marker gene of claim 1, said detection primer having the following sequence:
ADARB2 gene:
the corresponding detection primer of chr10: 1779052-1779183 is SEQ ID NO. 1-2;
the corresponding detection primer of chr10: 1779756-1779910 is SEQ ID NO 3-4;
ADCYAP1 Gene:
The corresponding detection primer of chr18:907522-907672 is SEQ ID NO. 5-6;
the corresponding detection primer of chr18:908206-908385 is SEQ ID NO 7-8;
ADHFE1 Gene:
The corresponding detection primer of chr8: 67344466-67344599 is SEQ ID NO 9-10;
C17orf64 gene:
the corresponding detection primer of chr17: 58498643-58498859 is SEQ ID NO. 11-12;
The corresponding detection primer of chr17: 58498879-58499081 is SEQ ID NO. 13-14;
CDO1 gene:
The corresponding detection primer of chr5: 115152259-115152414 is SEQ ID NO. 15-16;
GAD2 gene:
the corresponding detection primer of chr10: 26504914-26505063 is SEQ ID NO. 17-18;
the corresponding detection primer of chr10: 26506264-26506424 is SEQ ID NO. 19-20;
The corresponding detection primer of chr10: 26506907-26507068 is SEQ ID NO. 21-22;
GRM7 gene:
the corresponding detection primer of chr3: 6904068-6904274 is SEQ ID NO. 23-24;
GYPC gene:
The corresponding detection primer of chr2: 127413900-127414152 is SEQ ID NO. 25-26;
KCNA1 gene:
The corresponding detection primer of chr12: 5018675-5018893 is SEQ ID NO 27-28;
the corresponding detection primer of chr12: 5019386-5019578 is SEQ ID NO. 29-30;
KCNQ5 gene:
The corresponding detection primer of chr6: 73331034-73331188 is SEQ ID NO. 31-32;
The corresponding detection primer of chr6: 73331252-73331411 is SEQ ID NO. 33-34;
NETO1 Gene:
The corresponding detection primer of chr18: 70535858-70535996 is SEQ ID NO. 35-36;
RYR2 Gene:
the corresponding detection primer of chr1: 237205894-237206034 is SEQ ID NO. 37-38;
SORCS3 Gene:
The corresponding detection primer of chr10: 106400776-106400914 is SEQ ID NO 39-40;
the corresponding detection primer of chr10: 106401432-106401587 is SEQ ID NO. 41-42;
STX16 Gene:
The corresponding detection primer of chr20: 57225138-57225270 is SEQ ID NO. 43-44;
TCTEX1D1 Gene:
The corresponding detection primer of chr1: 67218010-67218198 is SEQ ID NO. 45-46;
TMEM101 gene:
The corresponding detection primer of chr17: 42092213-42092341 is SEQ ID NO. 47-48;
ZSCAN12 gene:
the corresponding detection primer of chr6: 28367209-28367379 is SEQ ID NO. 49-50;
the corresponding detection primer of chr6: 28367431-28367676 is SEQ ID NO. 51-52.
3. The use of a reagent for detecting a methylation region of a marker gene in the preparation of a detection kit or device, characterized in that the detection kit or device is used for detecting, screening or diagnosing endometrial cancer; the marker gene methylation region is the marker gene methylation region of claim 1.
4. A use according to claim 3, characterized in that: the reagent comprises at least one of an antibody, a probe, a primer and a mass spectrometry detection reagent which are specifically detected against the methylation region of the marker gene, wherein the primer comprises the detection primer of claim 2.
5. Use according to claim 3 or 4, characterized in that: the detection sample of the detection kit or the detection device comprises at least one of a body fluid sample, an exfoliated cell collection sample and a tumor tissue sample.
6. The use according to claim 5, characterized in that: the body fluid sample includes at least one of urine and blood; the exfoliated cell collection sample comprises at least one of a swab, a brush piece and a lavage fluid.
7. A multiplex PCR detection kit for early screening diagnosis of endometrial cancer, comprising reagents for detecting the methylation region of the marker gene of claim 1;
the reagent comprises at least one of an antibody, a probe, a primer and a mass spectrometry detection reagent which are specifically detected against the methylation region of the marker gene, wherein the primer comprises the detection primer of claim 2.
8. Use of the methylated gene marker according to claim 1 in the preparation of a diagnostic product for endometrial cancer.
9. Use of the multiplex PCR detection kit as claimed in claim 7 for the preparation of a diagnostic product for endometrial cancer.
10. Use according to claim 8 or 9, characterized in that: the diagnostic product includes any one of a kit, a formulation, and a chip.
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