RU2760573C1 - Set of oligonucleotide primers and fluorescently labelled probes for identifying the epigenetic markers of cervical cancer - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of medical biotechnology. In order to solve the posed problem, methylated RCGY sites of the promoter areas of the tumour marker genes CADM1, MAL, PAX1, DAPK1, FAM19A4, PRDM14 are determined using the GLAD-PCR method, and specially developed oligonucleotide primers and probes are used to determine these sections.

EFFECT: creation of a set of oligonucleotide primers and fluorescently labelled probes for determining the level of methylation of epigenetic markers causing the occurrence of cervical cancer by the GLAD-PCR method.

1 cl, 3 tbl, 1 dwg

Description

The invention relates to medicine, molecular biological research in the field of detection of oncological diseases and can be used for early non-invasive diagnosis of cervical cancer.

Cervical cancer (hereinafter - cervical cancer) is one of the most common cancers in women worldwide and ranks 4th in mortality. According to the WHO, almost every second woman in the world dies from cervical cancer. In the Russian Federation, 22 new cases of the disease are annually recorded for every 100,000 women, while every second one has precancerous conditions (HSIL). Modern methods make it possible to diagnose the disease in 78% of cases at only 3 stages.

Early detection of cervical cancer, in accordance with the Order of the Ministry of Health of the Russian Federation dated 04.24.2018 No. 186 "On approval of the Concept of predictive, preventive and personalized medicine", will accelerate the transition to personalized medicine and form an individualized approach to the treatment of the disease.

The current state of the screening program currently existing in the Russian Federation, which is included in the medical examination, shows insufficient effectiveness in detecting cervical cancer. There are two screening systems - organized (systematic) and unorganized (sporadic). Both systems contain the following diagnostic tools: cytological studies (including PAP test), PCR analysis for the presence of human papillomavirus of high oncogenic risk, ELISA analysis for tumor markers, colposcopy and biopsy. These research methods have a number of significant drawbacks - subjectivity in assessing the results, high qualifications of a doctor and laboratory assistant are required, as well as the impossibility of diagnosing malignant lesions localized in the cervical canal, which make up most of the pathologies of the cervix, technical errors associated with a violation of the technique for obtaining smears; all this reduces the level of effectiveness of screening programs. There may also be cases of discrepancy between the results of a cytological study and targeted biopsy, due to errors in the sampling of the material (volume, taking a biopsy from the wrong zone), endocervical location of the affected epithelium.

Directly, the diagnosis of cervical cancer is made only after the histological examination of biopsies, because carrying out cytological and biochemical studies is not enough for verification.

A biopsy is an invasive research method and a rather unpleasant and painful procedure that can only be performed by an experienced gynecologist, therefore, histological examination cannot be used as a screening study.

In recent years, a lot of studies have been carried out in the Russian Federation devoted to the diagnosis of cervical cancer using molecular genetic methods, despite this there are no kits for molecular genetic studies for the early diagnosis of cervical cancer.

Among the many studies related to the use of genetic markers of malignant tumors, the most urgent is the determination of the methylation level of individual genes. Gene methylation is a modification of a DNA molecule without changing the nucleotide sequence by attaching a methyl group to the cytosine in the CpG dinucleotide at the C5 position of the cytosine ring. Methylation in the promoter region of a gene leads to the suppression of the corresponding gene. Changes in gene expression as a result of their methylation can lead to the development of oncological diseases. In such cases, in tumor cells, total DNA hypomethylation is combined with local DNA hypermethylation of gene promoters associated with the regulation of the cell cycle and causing genome instability [Zaletaev D.V. et al. // Ecological genetics. - 2011. - T. 9. - No. 3; Tatarkova E.A. // Bulletin of the Adyghe State University. Series 4: Natural-mathematical and technical sciences. - 2014].

The level of gene methylation can be determined in different ways, based on the use of chemical modification of cytosine bases, methyl-sensitive restrictases, immunoprecipitation of methylated DNA, as well as various combinations of the above approaches [Skryabin N.A. et al. // Siberian Journal of Oncology. - 2013. - No. 6].

The most promising method for determining the presence of methylated DNA regions is a method based on the use of site-specific methyl-dependent endonucleases, which recognize only methylated DNA sequences.

The method based on the coincidence of the DNA sequence, which is the product of de novo methylation and the substrate specificity of the GlaI enzyme and which allows to determine the presence and number of copies of the 5'-R (5mC) GY-3 'site in the studied point of the genome, is called GLAD-PCR (GlaI hydrolysis and Ligation Adapter Dependent PCR) [Kuznetsov V.V. et al. Method for determining the nucleotide sequence of Pu (5mC) GPu at a given position of extended DNA // RF Patent No. 2525710 C1, publ. 08/20/2014, Bul. No. 23]. This method consists of three sequential stages - hydrolysis of the analyzed DNA using GlaI, ligation of a universal adapter, and real-time PCR reaction and is a universal method for determining the methylation level of R (5mC) GY sites, which makes it possible to form and describe a specific panel of genes specific for various types of cancer, but at the same time in the description of the method there are no gene panels specific for various types of oncology.

In recent decades, the number of studies devoted to the study of the level of methylation of genes of the promoter regions of genes-tumor markers of cervical cancer has increased. In 2018, the GYN-TEST was developed, based on the determination of the methylation level of a panel of genes ( ASTN1, DLX1, ITGA4, RXFP3, SOX17 and ZNF671 ) using bisulfite treatment without a DNA extraction procedure. The biological material used in the analysis is scrapings from the cervical canal [https://www.oncgnostics.com/gyntect-cervical-cancer/information-for-diagnostics-laboratories/test-principle/?lang=en].

Qiagen has a QIAsure Methylation Test Kit (FAM19A4 / mir124-2 DNA methylation test) for commercial use, aimed at determining the methylation level of the promoter regions of the FAM19A4 and mir124-2 genes in women with positive HPV status. The studied biomaterial is scrapings from the cervical canal.

The disadvantage of the above known analogs is the use of the bisulfite conversion of DNA method, which is characterized by laboriousness, high cost of research, significant time costs, DNA degradation and significant DNA loss, which leads to a decrease in accuracy and sensitivity.

The result of our invention is the creation of a set of oligonucleotide primers and fluorescently labeled probes for determining the level of methylation of epigenetic markers that cause cervical cancer by the GLAD-PCR method.

The specified technical result is achieved by solving the following tasks:

- carrying out bioinformatic analysis in order to select epigenetic markers of cervical cancer,

- creation of a set of oligonucleotide primers and fluorescently labeled probes to determine the methylation level of promoter regions of epigenetic markers using the GLAD-PCR method, the analysis results are taken into account by the method of hybridization-fluorescence detection of PCR amplification products in real time.

The essence of the invention is illustrated by the following examples

Example 1. Development of specific primers and fluorescently labeled probes for the CpG regions - islets of promoter regions of tumor marker genes and analysis by GLAD-PCR to determine the methylation level of promoter regions of tumor marker genes.

The promoter regions of the CADM1, MAL, PAX1, DAPK1, FAM19A4, and PRDM14 genes have been proposed as epigenetic markers. analysis of the level of hypermethylation of the regions of these genes makes it possible to determine both precancerous conditions and cancer at different stages of development [Luttmer R., et al. // British journal of cancer. - 2016. - Vol.115; Feng C., et al. // International Journal o Genmics. - 2018; Wang X., et al. // Frontiers Genetics. - 2018. - Vol. 9; Fang C., et al. // Molecular Genetics and Genomic Medicine. - 2019. - Vol. 7. - Issue 3; Dankai W., et al. // Plos One. - 2019] due to which early diagnosis of cervical cancer (stage 1-2) will be possible.

Bioinformatic analysis of tumor marker genes CADM1, MAL, PAX1, DAPK1, FAM19A4, and PRDM14 showed the presence of CpG islands in their promoter region, which contain several RCGY sites, the methylation of which can suppress gene expression in cervical cancer. In determining the methylated regions of epigenetic markers, we used the resources of the NCBI, as well as domestic and foreign publications available for analysis. As a result, the following fragments were selected that satisfy the specificity conditions for further preparation of primers (Table 1).

Table 1. Selected gene fragments for GLAD-PCR analysis

N Gene Sequence
RCGY site Coordinate
site start
RCGY one CADM1 GCGT 115504701 GCGT 115504984 2 MAL ACGC 95025112 ACGC 95025785 3 PAX1 GCGC 21706254 4 DAPK1 GCGC 87497727 5 TAFA4 (FAM19A4) GCGC 68932498 GCGT 68932948 6 PRDM14 ACGC 70069776

The selection of the structures of primers and fluorescent probes for the analysis of methylation of the selected sites was carried out using the Oligo 4.0 software. Oligonucleotide structures were selected according to the reference sequence of the human genome (assembly GRCh38.p7). Since the genomes of real people differ from the reference ("averaged") sequence, in order to minimize the likelihood of mismatch between the selected structures and the studied DNA samples, they were additionally checked against the dbSNP database for the content of frequent single nucleotide substitutions. At the same time, a frequency of no more than 1% was considered to be the permissible content of single nucleotide substitutions in the selected structures with a single substitution in each oligonucleotide structure. Each of the structures was also checked for uniqueness in the human genome using the BLAST web service (https://blast.ncbi.nlm.nih.gov).

To select a hybrid primer, the methodology was used, as well as the structure of the oligonucleotide adapter and hybrid primers according to the known method [Kuznetsov V.V. et al. Method for determining the nucleotide sequence of Pu (5mC) Gpu in a given position of an extended DNA sequence // RF Patent No. 2525710 C1, publ. 08/20/2014, Bul. No. 23].

The structure of the hybrid primer is a 19-nt sequence of the following composition: 5'-CCTGCTCTTTCATCCgYNN-3 ', where the 15-nt part of the primer complementary to the universal oligonucleotide adapter is underlined, and gYNN is the region complementary to the genomic sequence at the endolysis point of DNA methylase-dependent Gla. This structure assumes the presence of 32 variants of hybrid primers corresponding to different points of DNA hydrolysis at the R (5mC) GY site.

The sequences of the selected primers and fluorescent probes are shown in Table 2.

Table 2. Sequences of oligonucleotide primers and fluorescently labeled probes

Gene Sequences of fluorescent probe and genomic
primer (length in nucleotides) CADM1 SEQ ID NO: 1
SEQ ID NO: 2
SEQ ID NO: 3 5 'CCTGCTCTTTCATCGGTGC 3' (19)
5 'FAM-CCCGCCCCCTGGAGCCCG-BHQ1 3' (18)
5 'CGGTCTGATACAGCGATTGCTATAAACAT 3' (29) MAL SEQ ID NO: 4
SEQ ID NO: 5
SEQ ID NO: 6 5 'CCGACCTGCCCCAGCACC 3' (18)
5 'FAM-CCCAGCTGTGCCGGCCCCA-BHQ1 3' (19)
5 'CCTGCTCTTTCATCGGTCT 3' (19) PAX1 SEQ ID NO: 7
SEQ ID NO: 8
SEQ ID NO: 9 5 'CCTGCTCTTTCATCGGCCC 3' (19)
5 'FAM-TGCCCACTCCAAGCCAGACCCAGG-BHQ1 3' (24)
5 'TGGGTCCCCAGACAGCTCGG 3' (20) DAPK1 SEQ ID NO: 10
SEQ ID NO: 11
SEQ ID NO: 12 5 'CCTGCTCTTTCATCGGCAG 3' (19)
5 'FAM-CGGCGCGGCCCACCCACC-BHQ1 3' (18)
5 'GCGGCCAATGGGGACCCT 3' (18) TAFA4 (FAM19A4) SEQ ID NO: 13
SEQ ID NO: 14
SEQ ID NO: 15 5 'CCTGCTCTTTCATCGGTGT 3' (19)
5 'FAM-ACGTGGGCCGAGTTTTCTCCTCTCTCA-BHQ1 3' (27)
5 'GCCCGGCCCCAGCTGTATT 3' (19) PRDM14 SEQ ID NO: 16
SEQ ID NO: 17
SEQ ID NO: 18 5 'FAM-CCAGGTTCTGCGGGCTGCTCTCC-BHQ1 3' (23)
5 'CGGTCCCGGGATGGCTCT 3' (18)
5 'CCTGCTCTTTCATCGGCGG 3' (19)

The specificity of all selected primers was confirmed by testing in Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov). The melting point and the likelihood of dimer formation between oligonucleotides were tested in OligoAnalyzer 3.1 (https://eu.idtdna.com/calc/analyzer).

The course of work using a set of synthetic oligonucleotides and fluorescently labeled probes to determine the level of methylation of promoter DNA regions consists of the following steps:

1. Sampling and preparation of biological material - cervical scrapings;

2. Isolation of DNA from biological samples using a kit for DNA isolation (for example, "Proba-NK", manufacturer "DNA-Technology");

DNA preparations should have a purity of A260 / 280 from 1.4 to 1.8; A260 / 230 from 0.4 to 0.6, the concentration of nucleic acids should be from 80 to 250 ng / μl;

3. Analysis to determine the level of methylation of the promoter regions of the selected tumor marker genes using a set for GLAD-PCR analysis (manufactured by Sibenzaim LLC);

3.1. DNA preparation and hydrolysis includes preparation of PCR tubes (200 μl) according to the number of analyzed samples; mixing in each tube 14 μl of H ₂ O and 1 μl of isolated DNA; preparation of the general reaction mixture for DNA hydrolysis (H ₂ O, 10x SE TMN buffer, DMSO, BSA); adding the mixture to tubes with DNA samples and subsequent incubation for 30 minutes at 30 ° C;

3.2. Ligation (stitching) of DNA fragments with a universal adapter using DNA ligase includes preparation of a general reaction mixture for DNA hydrolysis (H ₂ O, universal adapter, ATP, T4 DNA ligase); adding this mixture to each tube and subsequent incubation for 15 minutes. at 25 ° C;

3.3. Real-time PCR includes the preparation of a PCR mixture for the studied site R (5mC) GY (H ₂ O, 10x SE GLAD buffer, MgCl ₂ , a mixture of triphosphates, BSA, a mixture of primers and SP Taq DNA Polymerase); adding the prepared PCR mixture to tubes with DNA; transfer of the resulting reaction mixture in a volume of 20 μl into the wells on a PCR plate; centrifuging the plate and placing it in a thermocycler for real-time PCR according to the following scheme:

- 3 min. at 95 ° C;

- 5 "blind" cycles: 95 ° C - 10 sec., 63 ° C - 20 sec., 72 ° C - 5 sec .;

- 40 cycles: 95 ° C - 10 sec., 63 ° C - 20 sec. (with fluorescent signal detection by FAM channel), 72 ° C - 5 sec .;

4. Determination of Cq values for all analyzed samples, by which the copy number is established and the percentage of methylation of the selected sites specific for cervical cancer is determined (Fig. 1).

Example 2. GLAD-PCR analysis for determining the methylation level of tumor marker genes for cervical cancer

For GLAD-PCR analysis, we used samples of cervical scrapings obtained from patients with cervical cancer (n = 12) and healthy people (n = 5). DNA was isolated from the samples using the "Extraction 100" kit (manufacturer "Vector-Best", Russia). The concentration of the samples was determined using a Nano Drop 2000 spectrophotometer.

The isolated DNA preparations had a purity of A260 / 280 from 1.4 to 1.8; A260 / 230 from 0.4 to 0.6. Nucleic acid concentrations ranged from 80 to 250 ng / μl.

The GLAD-PCR reaction was performed as described in Example 1. Hela genomic DNA was used as a positive control.

The results are shown in Table 3.

Table 3. Results of analysis of cervical scrapings samples for the presence of methylation of promoter regions of tumor marker genes

Sample no. Diagnosis* CADM1 MAL PAX1 DAPK1 TAFA4 (FAM19A4) PRDM14 one RShM + + + + + + 2 RShM + - + + + + 3 RShM + + + + + - 4 RShM + + + + + + 5 RShM + + - + + + 6 RShM + + + - + + 7 RShM + + + + + + eight RShM + + + + + + 9 RShM + - + + + + 10 RShM + + + - + + eleven RShM + + + + + + 12 RShM + + + + + + thirteen Norm (no pathologies) - - - - - - 14 Norm (no pathologies) - - - - - - 15 Norm (no pathologies) - - - - - - sixteen Norm (no pathologies) - - - - - - 17 Norm (no pathologies) - - - - - - Number (%) of identified cervical cancer specimens 12 (100%) 12 (100%) 10 (83.3%) 11 (91.6%) 10 (83.3%) 12 (100%) 11 (91.6%) Hela, genomic DNA + + + + + +

Note: "cervical cancer" - cervical cancer; "*" - the diagnosis is made on the basis of the histological conclusion; "+" - the presence of methylated sites in the promoter region of genes.

The analyzed sample is considered positive when the fluorescence accumulation curve had a characteristic exponential growth phase and the Ct value is less than or equal to 35, which means the presence of methylated sites in the promoter region of tumor marker genes. The analyzed sample is considered negative when there is no fluorescence accumulation curve, or has a Ct value greater than or equal to 35 - there are no methylated sites in the promoter region.

The presence or absence of methylated sites in epigenetic markers of cervical cancer gives an idea of the gene methylation status and can be used as an additional tool for diagnosing cervical cancer in the early stages.

The use of the proposed solution allows you to achieve the technical result: the creation of a set of oligonucleotide primers and fluorescently labeled probes to determine the level of methylation of epigenetic markers causing cervical cancer by the GLAD-PCR method.

The main advantages of the proposed method are:

• use of available PCR equipment,

• analysis time - 2-4 hours;

• ease of use - 3 consecutive steps in one tube;

• high sensitivity - DNA loss is minimal;

• high specificity - due to the use of highly specific primers and probes.

The technical result was achieved by carrying out bioinformatic analysis in order to select epigenetic markers of cervical cancer and create a set of oligonucleotide primers and fluorescently labeled probes to determine the methylation level of promoter sites of epigenetic markers using the GLAD-PCR method, the analysis results are taken into account by the method of hybridization-fluorescence detection products. amplification in real time.

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LIST OF SEQUENCES

<110> Es G Limited Liability Company

<120> Set of oligonucleotide primers and fluorescently labeled

probes to detect epigenetic markers of cervical cancer.

<212> DNA

<213> Homo sapiens

<223> for specific primers and probes to the regions of CpG islands

promoter regions of CADM1 tumor marker genes

<210> 1

<211> 19

<400> 5'-CCTGCTCTTTCATCGGTGC-3 '

<210> 2

<211> 18

<400> 5'-FAM-CCCGCCCCCTGGAGCCCG-BHQ1-3 '

<210> 3

<211> 29

<400> 5'-CGGTCTGATACAGCGATTGCTATAAACAT-3 '

<223> for specific primers and probes to the regions of CpG islands

promoter regions of MAL tumor marker genes

<210> 4

<211> 18

<400> 5'-CCGACCTGCCCCAGCACC-3 '

<210> 5

<211> 19

<400> 5'-FAM-CCCAGCTGTGCCGGCCCCA-BHQ1-3 '

<210> 6

<211> 19

<400> 5'-CCTGCTCTTTCATCGGTCT-3 '

<223> for specific primers and probes to the regions of CpG islands

promoter regions of PAX1 tumor marker genes

<210> 7

<211> 19

<400> 5'-CCTGCTCTTTCATCGGCCC-3 '

<210> 8

<211> 24

<400> 5'-FAM-TGCCCACTCCAAGCCAGACCCAGG-BHQ1-3 '

<210> 9

<211> 20

<400> 5'-TGGGTCCCCAGACAGCTCGG-3 '

<223> for specific primers and probes to the regions of CpG islands

promoter regions of DAPK1 tumor marker genes

<210> 10

<211> 19

<400> 5'-CCTGCTCTTTCATCGGCAG-3 '

<210> 11

<211> 18

<400> 5'-FAM-CGGCGCGGCCCACCCACC-BHQ1-3 '(18 n)

<210> 12

<211> 18

<400> 5'-GCGGCCAATGGGGACCCT-3 '(18 n)

<223> for specific primers and probes to CpG island regions

promoter regions of TAFA4 tumor marker genes (FAM19A4)

<210> 13

<211> 19

<400> 5'-CCTGCTCTTTCATCGGTGT-3 '

<210> 14

<211> 27

<400> 5'-FAM-ACGTGGGCCGAGTTTTCTCCTCTCTCA-BHQ1-3 '

<210> 15

<211> 19

<400> 5'-GCCCGGCCCCAGCTGTATT-3 '

<223> for specific primers and probes per site

CpG islands of promoter regions of PRDM14 tumor marker genes

<210> 16

<211> 16

<400> 5'-FAM-CCAGGTTCTGCGGGCTGCTCTCC-BHQ1-3 '(23 n)

<210> 17

<211> 18

<400> 5'-CGGTCCCGGGATGGCTCT-3 '

<210> 18

<211> 19

<400> 5'-CCTGCTCTTTCATCGGCGG-3 '

<---

Claims (2)

A set of oligonucleotide primers and fluorescently labeled probes for determining the level of methylation of promoter regions of tumor marker genes for early non-invasive diagnosis of cervical cancer using GLAD-PCR analysis, in DNA samples with the following structure: Gene Sequences of fluorescent probe and genomic
primer (length in nucleotides) CADM1 SEQ ID NO: 1
SEQ ID NO: 2
SEQ ID NO: 3 5 'CCTGCTCTTTCATCGGTGC 3' (19)
5 'FAM-CCCGCCCCCTGGAGCCCG-BHQ1 3' (18)
5 'CGGTCTGATACAGCGATTGCTATAAACAT 3' (29) MAL SEQ ID NO: 4
SEQ ID NO: 5
SEQ ID NO: 6 5 'CCGACCTGCCCCAGCACC 3' (18)
5 'FAM-CCCAGCTGTGCCGGCCCCA-BHQ1 3' (19)
5 'CCTGCTCTTTCATCGGTCT 3' (19) PAX1 SEQ ID NO: 7
SEQ ID NO: 8
SEQ ID NO: 9 5 'CCTGCTCTTTCATCGGCCC 3' (19)
5 'FAM-TGCCCACTCCAAGCCAGACCCAGG-BHQ1 3' (24)
5 'TGGGTCCCCAGACAGCTCGG 3' (20) DAPK1 SEQ ID NO: 10
SEQ ID NO: 11
SEQ ID NO: 12 5 'CCTGCTCTTTCATCGGCAG 3' (19)
5 'FAM-CGGCGCGGCCCACCCACC-BHQ1 3' (18)
5 'GCGGCCAATGGGGACCCT 3' (18) TAFA4 (FAM19A4) SEQ ID NO: 13
SEQ ID NO: 14
SEQ ID NO: 15 5 'CCTGCTCTTTCATCGGTGT 3' (19)
5 'FAM-ACGTGGGCCGAGTTTTCTCCTCTCTCA-BHQ1 3' (27)
5 'GCCCGGCCCCAGCTGTATT 3' (19) PRDM14 SEQ ID NO: 16
SEQ ID NO: 17
SEQ ID NO: 18 5 'FAM-CCAGGTTCTGCGGGCTGCTCTCC-BHQ1 3' (23)
5 'CGGTCCCGGGATGGCTCT 3' (18)
5 'CCTGCTCTTTCATCGGCGG 3' (19)

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