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CN-121992103-A - Methylation marker combination for early screening of endometrial cancer

CN121992103ACN 121992103 ACN121992103 ACN 121992103ACN-121992103-A

Abstract

The invention discloses a methylation marker combination for early screening of endometrial cancer, belonging to the technical field of molecular diagnosis and bioinformatics intersection. Three methylation markers of CDO1, NKX2-6 and SORCS are screened out through bioinformatics analysis, NGS targeted sequencing and qPCR verification, specific primers SEQ ID NO.1-2, 4-5 and 7-8 and probes SEQ ID NO.3, 6 and 9 are designed, and a fluorescent quantitative PCR detection system is constructed by combining internal reference genes BACT. According to the method, through detecting the methylation level of the three gene promoter regions in the sample, the cutoff value is 0.36, the sensitivity of endometrial cancer diagnosis of the exfoliated cells is 90.37%, the specificity is 89.60%, and the AUC= 0.9682 can effectively distinguish benign samples from cancerous samples. The invention realizes noninvasive/minimally invasive early screening of endometrial cancer, improves diagnosis accuracy and safety, wherein NKX2-6 and SORCS are novel discovery markers, and have important scientific value and clinical transformation potential.

Inventors

  • LIU AIJUN
  • LIN YANDIE
  • ZHENG HAONING
  • LI JING
  • SHAO LIWEI
  • GU CHENGLEI
  • MA ZIYU

Assignees

  • 中国人民解放军总医院第七医学中心

Dates

Publication Date
20260508
Application Date
20260211

Claims (9)

  1. 1. A methylation marker combination for early screening of endometrial cancer, wherein the marker combination is CDO1, NKX2-6 and SORCS genes.
  2. 2. The primer and probe for detecting the methylation marker combination of claim 1 are characterized in that the sequences of the primer and probe for detecting the methylation of the CDO1 gene are shown in SEQ ID NO. 1-3, the sequences of the primer and probe for detecting the methylation of the NKX2-6 gene are shown in SEQ ID NO. 4-6, and the sequences of the primer and probe for detecting the methylation of the SORCS gene are shown in SEQ ID NO. 7-9.
  3. 3. The primer and probe of claim 2, wherein the probes of SEQ ID No.3, SEQ ID No.6, SEQ ID No.9 are 5 'labeled with FAM fluorescent reporter groups and 3' labeled with MGB quencher groups.
  4. 4. Use of the primers and probes of claim 2 or 3 for the preparation of an endometrial cancer early screening reagent or kit.
  5. 5. A reagent or kit for early screening of endometrial cancer, wherein the reagent or kit comprises the primer and probe of claim 2 or 3.
  6. 6. The reagent or kit according to claim 5, wherein the reagent or kit further comprises a primer and a probe for detecting the reference gene BACT, and the sequences of the primer and the probe for detecting the reference gene BACT are shown in SEQ ID NO. 10-12.
  7. 7. The reagent or kit according to claim 6, wherein the reagent or kit further comprises bisulphite.
  8. 8. The reagent or kit according to claim 7, wherein the bisulphite is sodium bisulphite.
  9. 9. A system for diagnosing endometrial cancer, wherein the system is a computing device that concludes a diagnosis based on the detection of the methylation marker of claim 1 in a sample of a subject, the system comprising: (1) Sample collection and processing device for collecting sample from subject and processing the sample; (2) Methylation detection means; (3) And a calculation device for obtaining a diagnosis conclusion according to the detection result of the methylation marker of the subject sample.

Description

Methylation marker combination for early screening of endometrial cancer Technical Field The invention belongs to the technical field of molecular diagnosis and bioinformatics intersection, and particularly relates to a methylation marker combination for early screening of endometrial cancer. Background Endometrial cancer (Endometrial Carcinoma, EC) is one of the three major malignancies of the female reproductive system, and its global disease burden presents a significant upward trend. The global new occurrence rate is increased by 132% and the death rate is increased by 98% in the past thirty years, and the rapid rising trend is closely related to population aging, obesity rate increase and metabolic syndrome epidemic. Endometrial cancer is a highly malignant tumor of female reproductive system, and early diagnosis is closely related to prognosis improvement. It is counted that the 5-year survival rate of patients in stage I can reach more than 95%, and the survival rate suddenly drops to 17% -45% when the patients progress to stages III-IV. This significant survival difference highlights the clinical value of accurate early diagnosis techniques. The current diagnostic procedures used clinically rely mainly on a combination of imaging and invasive examination. Transvaginal Ultrasound (TUVs) is the method of choice for early screening and diagnosis in the clinic. As a first-line screening tool, its diagnostic efficacy is significantly disturbed by physiological proliferation. A large cohort of more than one thousand people shows that when the endometrial thickness is more than or equal to 5mm as a diagnostic threshold for postmenopausal women, although the sensitivity is as high as 96.2% and the negative predictive value is as high as 99.3%, the specificity is only 51.5%, resulting in the need for unnecessary invasive examination of nearly half of the patients. The hysteroscope can directly sample suspicious lesions and is suitable for patients with TUV detection positive or repeated symptoms, and is a gold standard for diagnosing endometrial cancer. Information obtained from endometrial biopsies is often used for pre-operative disease staging, which is critical to the scope of surgery management and guidance. Hysteroscopy, however, also increases the risk of cancer spread, and physical discomfort and false negative results are drawbacks of this approach. Therefore, there is a need to develop safer, more efficient and reliable screening and early diagnosis means in high risk populations to reduce the mortality of endometrial cancer and improve clinical prognosis. In recent years, the breakthrough of molecular diagnosis technology and the application of DNA methylation in early diagnosis of cancer provide a new direction for early detection of endometrial cancer. DNA methylation abnormality mainly occurs in CpG islands in gene promoter regions, and the DNA methylation abnormality is involved in tumor occurrence and development by regulating and controlling the expression of tumor key genes. As a stable epigenetic modification, DNA methylation can undergo characteristic changes in the early stages of endometrial carcinogenesis, making it an ideal early marker. Currently, there are various methods for detecting DNA methylation, including whole genome methylation detection techniques (WGBS, RRBS, illumina EPIC Bead Chip microarray, meDIP-seq, methylRAD) and site-specific methylation detection techniques (pyrosequencing, MASSARRAY, BSP, MSP). These mature detection techniques provide a powerful support for large-scale screening and validation of methylation markers. Therefore, the identification and optimization of a group of DNA methylation marker combinations with high sensitivity and high specificity in the early stage of EC has a great significance in developing an efficient noninvasive or minimally invasive early screening method for endometrial cancer, realizing accurate diagnosis and improving prognosis of patients. Disclosure of Invention The invention screens 184,915 differential methylation sites (DMP) and 1,215 Differential Methylation Regions (DMR) through bioinformatics analysis, defines the DMP and the DMR, and carries out gene annotation to obtain 145 differential methylation genes of promoters, and finally screens 10 genes which are possibly related to endometrial cancer process and show high methylation level according to GO and KEGG enrichment analysis. Designing methylation specific primer groups for CpG islands of the 10 gene promoter regions based on Ion AMPLISEQ DESIGNER platform. Targeted sequencing was performed on 50 benign endomembrane samples and 50 endometrial cancer samples using high throughput sequencing technology. The data analysis shows that the 5 genes CDO1, TRH, PENK, NKX-6 and SORCS are in a low methylation level state in benign endometrial cancer samples, the average methylation frequency is lower than 5%, the average methylation frequency is higher than 20% in endometrial