RU2753002C1 - Method for determining genetic markers for assessing polygenic risk of developing hormone-positive subtype of breast cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of personalized medicine and concerns a method for determining genetic markers of polygenic risk of breast cancer (BC). A method for analyzing 30 genetic markers to assess the polygenic risk of developing a hormone-positive subtype of breast cancer is proposed: TGFBR2 (rs12493607), 18q11.2 (rs527616), 19p13 (rs2363956), 19p13 (rs8170), 22q12.2 (rs132390), 2q31.1 (rs2016394), 3p26.2 (rs6762644), 7q35 (rs720475), 8q24 (RS13281615), 9q31 (rs865686), ADAM29 (RS6828523), ANKRD16 (RS2380205), CCDC88C (RS941764), CCND1 (RS554219), CCND1 (RS75915166), CDYL2 (RS13329835), CHST9 (RS1436904), COX11 (RS6504950), DNAJC1 (rs7072776), LGR6 (rs6678914), MAP3K1 (rs889312), MDM4 (rs4245739), NRIP1 (rs2823093), PAX9 (rs2236007), RAD51L1 (rs2588809), RANBP1 (rs204247), SLC4A7 (rs4973768), TERT (rs10069690), TET2 (rs9790517), ZMIZ1 (rs704010). A set of oligonucleotide probes necessary for the identification of genetic markers of polygenic risk of developing a hormone-positive subtype of breast cancer is proposed.

EFFECT: invention provides for simultaneous determination of genotypes at selected loci using multiplex PCR and subsequent hybridization with an oligonucleotide biological microchip (biochip).

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Description

The technical field to which the invention relates

The invention relates to the field of personalized medicine and relates to a method for determining genetic markers of polygenic risk of development (PGR) of a hormone-positive subtype of breast cancer (BC).

One of the important risk factors for the development of breast cancer is a genetic predisposition. In addition to highly penetrant and medium penetrate mutations that cause hereditary forms of breast cancer, a number of single nucleotide polymorphisms (SNPs) have been identified, each of which has a small contribution to the risk of developing breast cancer, but their combined effect in assessing the polygenic risk turns out to be significant and can improve the stratification of the population by risk groups. The inclusion of informative genetic markers of GHR in the genetic analysis can significantly expand the possibilities of identifying a predisposition to breast cancer and will allow targeted implementation of measures aimed at prevention and early diagnosis.

There are hormone-negative and hormone-positive subtypes of breast cancer, which differ in the mechanisms of occurrence, genetic characteristics, the nature of the course and the therapy used. The most common is hormone-positive breast cancer. Many known markers of GHR are informative for this particular subtype of the disease.

This invention is intended to identify genetic markers that can be used to assess the GHR hormone-positive subtype of breast cancer.

State of the art

Breast cancer (BC) is a multifactorial cancer. One of the important risk factors for the development of breast cancer is a genetic predisposition. The contribution of high and medium-risk mutations (mutations in the genes BRCA1, BRCA2, PALB2, NBN, BLM, ATM, RAD50 and some others) is only about 25% in the case of familial forms of breast cancer and about 5-10% in the general susceptibility to breast cancer.

When conducting genome wide association studies (GWAS) on large cohorts of breast cancer patients and healthy donors (tens of thousands of people each), many single nucleotide polymorphisms were identified in various genes and chromosomal loci associated with an increased risk of developing breast cancer. Each of the polymorphisms individually has an insignificant effect on the likelihood of developing the disease and cannot be considered as an independent risk factor. However, their combined effect can lead to a significant increase in the risk of developing the disease, which made it possible to develop polygenic risk models. With the accumulation of scientific information, models of PHR were proposed based on various combinations of polymorphisms in the amount of 4-10 SNPs [Reeves, Gillian K., et al. "Incidence of breast cancer and its subtypes in relation to individual and multiple low-penetrance genetic susceptibility loci." Jama 304.4 (2010): 426-434], 77 SNP [Mavaddat N. et al. Prediction of breast cancer risk based on profiling with common genetic variants // JNCI: Journal of the National Cancer Institute. - 2015. - T. 107. - No. 5], 88 SNP [Kuchenbaecker K. B. et al. Evaluation of polygenic risk scores for breast and ovarian cancer risk prediction in BRCA1 and BRCA2 mutation carriers // JNCI: Journal of the National Cancer Institute. - 2017. - T. 109. - No. 7], 313 SNP [Mavaddat N. et al. Polygenic risk scores for prediction of breast cancer and breast cancer subtypes // The American Journal of Human Genetics. - 2019. - T. 104. -№. 1. - C. 21-34].

High-performance genotyping technologies, such as high-density biochips from Illumina, for example, the Oncoarray array (500,000 markers), were used to search for markers for PGR models. The iCOGSarray genotyping array (211,155 markers) was jointly developed by the COGS consortium and Illumina specifically to study the genetic markers of hormone-related cancers: breast cancer, ovarian cancer, prostate cancer [Bahcall O., Orli B. COGS project and design of the iCOGS array // Nat Genet. - 2013. - T. 45. - No. 4. - P. 343]. The approaches based on high-throughput genotyping techniques are highly informative and have allowed the development of models for calculating polygenic risk, where each marker is assigned a coefficient according to its contribution to PGR. When introducing into practice and testing the developed HGR models, the use of high-performance methods may turn out to be excessive in terms of the necessary costs (high cost of reagents and equipment, labor intensity and duration of analysis) and the fact that a small part of them is included in the analysis out of hundreds of thousands of markers. In a study on the development of a PGR model based on 14 markers known from the GWAS results [Reeves, Gillian K, et al. "Incidence of breast cancer and its subtypes in relation to individual and multiple low-penetrance genetic susceptibility loci." Jama 304.4 (2010): 426-434], real-time PCR with Taqman probes was used for genotyping. This method is fast and accurate, but it is not suitable for detecting a large number of markers at the same time. in one tube it is possible to analyze only 1-3 markers. The study of many models of PGR based on 70-80 markers was carried out with genotyping on high-density biochips Illumina [Shieh Y. et al. Breast cancer risk prediction using a clinical risk model and polygenic risk score // Breast cancer research and treatment. - 2016. -T. 159. - No. 3. - C. 513-525; Vachon S.M. et al. The contributions of breast density and common genetic variation to breast cancer risk // Journal of the National Cancer Institute. - 2015. - T. 107. - No. 5. - C. dju397; Dite G. S. et al. Breast cancer risk prediction using clinical models and 77 independent risk-associated SNPs for women aged under 50 years: Australian Breast Cancer Family Registry // Cancer Epidemiology and Prevention Biomarkers. - 2016. - T. 25. - No. 2. - C. 359-365].

Such time-consuming and expensive analyzes can be a serious obstacle when conducting similar studies in the Russian population, and even more so when introducing such testing into clinical practice. Thus, in this area there is an objective need for the development of effective and affordable tools for genetic testing in order to assess the polygenic risk of breast cancer, which would be advantageously distinguished by the simplicity of the analysis, high information content and low cost.

The method proposed in the present invention has a number of important advantages: fast and simple sample preparation, low cost of reagents and necessary equipment, the ability to simultaneously determine a wide range of genetic markers. This method concerns the determination of genetic markers of hormone-positive breast cancer.

Disclosure of the essence of the invention

The objective of the present invention is to provide a method for determining genetic markers of the polygenic risk of developing a hormone-positive subtype of breast cancer. The panel includes 38 genetic markers that allow assessing the level of polygenic risk of breast cancer development.

The essence of the invention is to provide a method for analyzing 30 SNP (Table 1).

The main features of this invention are multiplex PCR and subsequent hybridization of products on a biochip containing a set of immobilized oligonucleotide probes.

The first important feature of the invention is a set of primers for amplification of the analyzed loci, which is used to obtain fluorescently labeled products in the required amount for hybridization on a biochip. The sequences of primers for PCR are shown in Sequence Listing 1 (SEQ ID NO: 1-60).

The second important feature of the invention is a biochip containing a set of immobilized oligonucleotide probes, the sequences of which are shown in Sequence Listing 2 (SEQ ID NO: 61-120). Oligonucleotide probes are immobilized in the cells of a hydrogel microchip, as described in patent RU 2206575 (publication date 2003-06-20) at a concentration of 2000 μM. The layout of the probes in the cells of the biochip is shown in Figure 1.

A set of PCR primers is used for multiplex PCR, which results in a fluorescently labeled product that is used for subsequent hybridization on a biochip. Next, hybridization of the PCR product with oligonucleotide probes immobilized in the cells of the biochip is carried out. After hybridization and washing of the biochip, the obtained fluorescence pattern is analyzed, on the basis of which a report on the genotype of the sample under study is made. An example of a hybridization pattern is shown in Figure 2.

Brief Description of Tables and Shapes

Figure 1. Scheme of the location of probes in the cells of the biochip to determine the genetic markers of the polygenic risk of the hormone-positive subtype of breast cancer (a) the location of the cells on the biochip; (b) explanation of symbols.

Figure 2. Hybridization pattern of the analysis of genetic markers of the polygenic risk of hormone-positive breast cancer subtype in a control DNA sample obtained on a biochip.

Table 1. Panel of detectable genetic markers.

Implementation of the invention

To ensure optimal conditions for multiplex PCR, it is necessary to use primers that provide specific and efficient annealing on the target at the same temperature, and they should not form secondary structures at this temperature, including under multiplex PCR conditions. For optimal (stable and specific) production of the amount of fluorescently labeled PCR product required for hybridization on a biochip, the concentration of reagents in the reaction mixture and the temperature-time regime of the reaction must be selected and optimized. As a result of the work carried out, primers and conditions for carrying out PCR were selected, which allow efficient production of the studied DNA loci under conditions of multiplex PCR.

For immobilization on a biochip, oligonucleotide probes are selected that allow identification of all gene regions selected for analysis.

Oligonucleotide probes should be selected according to the following criteria:

1) The oligonucleotide probe must have a high specificity for the analyzed locus.

2) Preferably, the location of the variable nucleotide in the middle region of the probe, since such a probe design allows for better discrimination between perfect and imperfect duplexes.

3) The oligonucleotide probe under the conditions under which the hybridization is carried out should not form stable secondary structures, the presence of which can lead to a decrease in the efficiency of hybridization.

To analyze the genetic markers of the polygenic risk of the hormone-positive subtype of breast cancer, 60 highly specific differentiating oligonucleotide probes (Sequence Listing 2, SEQ ID NOs: 61-120) were immobilized on the biochip, the structure of which provides highly specific binding to completely complementary DNA targets. A bright fluorescent signal is observed only in cells in which a perfect duplex was formed between the oligonucleotide probe and the fluorescently labeled PCR product during hybridization.

Here is the sequence of analysis using this method.

Amplification of target DNA regions to obtain predominantly single-stranded fluorescently labeled PCR products is carried out by means of multiplex PCR. For PCR, primers SEQ ID NO: 1-60 are used (The primer sequences are shown in Sequence Listing 1). A DNA sample isolated from blood or scraping of buccal epithelium by any method known to the researcher (phenol-chloroform extraction, isolation on columns, isolation on a sorbent, etc.) is used as a matrix for the reaction. Multiplex PCR can be performed in one or two rounds. When staged in two rounds, in the first round, a symmetrical production of the analyzed regions is carried out (forward and reverse primers in the same concentrations), in the second round (where the product of the 1st PCR stage is used as a matrix), reverse primers are added in excess, due to which single-stranded PCR product. For setting in one round, an additional region is introduced at the 5'-end of each reverse primer, the sequence of which corresponds to the sequence of the universal primer, which is added to the reaction in excess, and provides the synthesis of a predominantly single-chain product [Fesenko D.О. et al. Biochip-based genotyping assay for detection of polymorphisms in pigmentation genes associated with cutaneous melanoma // Genetic Testing and Molecular Biomarkers. - 2016. - T. 20. - No. 4. - C. 208-212]. In the course of multiplex PCR, fluorescent labeling of PCR products is carried out using dUTP-Cy5. Any fluorochrome without limitation (for example, FITC, Texas red, Su-3, etc.), as well as biotin, can also be used as a fluorescent label.

PCR can be performed using any kind of thermostable polymerase (Taq polymerase, Taq hot start polymerase, Tth polymerase, Tfl polymerase, Pfu polymerase, Vent polymerase, DeepVent polymerase, and other commercially available enzymes) working in corresponding buffer. To build a new chain, a mixture of dNTPs (dATP, dGTP, dCTP, dTTP) is added to the buffer. For PCR, ready-made commercially available kits containing all the necessary components, except for primers, can be used.

Fluorescently labeled DNA fragments obtained after PCR are hybridized with oligonucleotide probes immobilized in the gel cells, the sequences of which are regions complementary to the sequences of the analyzed regions.

Hybridization of the PCR product with oligonucleotide probes on the biochip can be carried out in any hybridization buffer, for example, in SSPE buffer with formamide or buffer with guanidine. Hybridization is carried out for 8-14 hours at 37 ° C. Washing the biochip after hybridization can be carried out in any buffer known in the art (SSC, SSPE, etc.) or in distilled water.

The analyzed DNA fragment under the conditions (composition of the reaction, temperature and time of hybridization) under which hybridization occurs, forms perfect duplexes only with oligonucleotide probes that are completely complementary to it. The fluorescence signal is detected only in cells in which a perfect duplex is formed. If the duplex is imperfect (at least one unpaired nucleotide is present), then there is no fluorescence signal.

Further, the results of hybridization are visualized using any detection system capable of exciting fluorescence and recognizing a fluorescent signal (for example, a portable biochip analyzer equipped with a CCD camera and special software manufactured by BIOCHIP-IMB LLC (Moscow, Russia)).

The production of biochips can be carried out by sequentially applying acrylamide gel cells to the surface of a glass substrate, activating the cells and immobilizing modified oligonucleotides in the cells [Analvsis of SNPs and other genomic variations using gel-based chips / A. Kolchinsky, A. Mirzabekov // Hum Mutat. - 2002. - Vol.19. - P. 343-360. Review]. In addition to glass, any material can be used as a substrate, including metal, flexible membranes and plastic (Patent RU 2309959, published 2007-11-10). Biochips can also be manufactured by other known methods [Arrays of immobilized oligonucleotides - contributions to nucleic acids technology / H. Seliger, M. Hinz, E. Happ // Curr Pharm Biotechnol-2003. - Vol. 4. - P. 379-395].

For the manufacture of a biochip in the present invention uses a set of oligonucleotide probes SEQ ID NO: 61-120, shown in Sequence Listing 2. The location of specific oligonucleotide probes on the biochip may vary depending on the ease of interpretation of the results.

The following are examples that illustrate the possibilities of using the method for determining genetic markers to assess the polygenic risk of developing a hormone-positive subtype of breast cancer. Variants and modifications of the invention, which can be reproduced without departing from the general concept of the present invention and without involving one's own inventive activity, will also fall within the scope of the present invention.

Example 1. Amplification of target DNA in order to obtain a fluorescently labeled PCR product in the required amount

DNA was isolated from blood using the QIAamp DNA Blood Mini Kit (Qiagen, USA).

The development of the regions of the analyzed genes was carried out by the method of one-stage multiplex PCR. The 5'-TCATTGGATCTCATTA-3 'sequence was introduced at the 5'-end of each reverse primer during synthesis. PCR was performed in two separate reactions for each DNA sample. PCR mixture with a total volume of 25 μl included: 0.5 units. Act. Hot start Taq polymerase SynTaq (Syntol, Russia), 1 × PCR buffer (Syntol, Russia), 200 μM each dNTP (SibEnzyme, Russia), 10 ng DNA, 50 pmol universal primer with the sequence 5'-TCATTGGATCTCATTA-3 ', 8 μM fluorescently labeled dUTP-Cy7, 1 pmol of each primer: reaction # 1 contained primers SEQ ID NO: 1-30, reaction # 2 contained primers SEQ ID NO: 31-60. Amplification was carried out according to the following scheme: 40 cycles (94 ° C 30s, 65 ° C 30s, 67 ° C 30s, 69 ° C 30s, 72 ° C 20s), then 40 cycles (94 ° C 30s, 56 ° C 10s, 72 ° С 30s), on a T100 amplifier (Bio-Rad, USA).

Example 2. Oligonucleotide biochip for the analysis of genetic markers of the polygenic risk of developing hormone-positive breast cancer subtype

Oligonucleotide probes for immobilization on a microchip were synthesized on a 394 DNA / RNA Synthesizer (Applied Biosystems, USA) using a standard phosphoamidite procedure. A spacer with a free amino group was introduced at the 3'-end of the oligonucleotides using the 3'-Amino-Modifier C7 CPG 500 method (Glen Research, USA).

The biochip was prepared by copolymerizing an oligonucleotide in an acrylamide gel (patent RU 2309959, published 2007-11-10 and RU 2175972, published 2001-11-20). The biochip contains 76 immobilized oligonucleotide probes (SEQ ID NO: 60-120), a list of which is also presented in Sequence Listing 2. The wells were applied according to the scheme in Figure 1.

Example 3. Hybridization of a fluorescently labeled product on a biochip

The reaction mixture obtained after the PCR described in Example 1 was used for hybridization on a biochip. 10 μl of formamide (Rapgeas, Spain), 10 μl of 20 × SSPE (Thermo Fisher Scientific, USA), 10 μl of PCR product from reaction No. 1 and 10 μl of PCR product from reaction No. 2 were mixed. The resulting mixture was applied to a biochip and left for 12 hours at 37 ° C. After hybridization, the biochip was washed in 1 × SSPE for 10 minutes at room temperature.

Example 4. Registration and interpretation of hybridization results

The hybridization pattern was recorded using a portable biochip analyzer equipped with a CCD camera manufactured by Biochip-IMB LLC. According to the obtained hybridization pattern shown in Figure 2, the genotype of the sample was determined Genotype of the sample:

1.RS12493607 SS

2.rs132390 TT

3.rs13281615 AG

4.rs13329835 AA

5.RS1436904 TT

6.RS2016394 TT

7.rs204247 AG

8.rs2236007 GG

9.rs2588809 CC

10.rs2823093 GG

11.rs4245739 AC

12.rs4973768 CT

13.rs527616 CC

14.rs554219 CC

15.rs6504950 GA

16.rs6678914 GG

17.rs6762644 AG

18.rs6828523 AA

19.rs704010 CT

20.rs7072776 GG

21. rs720475 GG

22. rs75915166 CC

23. rs865686 TT

24. rs889312 AC

25. rs941764 AG

26.rs9790517 CC

27. rs10069690 CC

28. rs2363956 GT

29. rs23 80205 CT

30. rs8170 GG

Claims (6)

1. A method for determining 30 genetic markers to assess the polygenic risk of developing a hormone-positive subtype of breast cancer (BC), which includes the following stages: (a) - amplification of the analyzed by means of multiplex PCR, in which DNA isolated from blood or other biomaterial obtained from a patient is used as a template for amplification, and primers represented by the sequences SEQ ID NO: 1-60; (b) - providing a biochip for the analysis and identification of genetic markers of the polygenic risk of developing hormone-positive breast cancer subtype, with a set of oligonucleotide probes represented by the sequences SEQ ID NO: 61-120; (c) - hybridization of the fluorescently labeled PCR product obtained in step (a) on the biochip obtained in step (b); (d) - registration and interpretation of the results of hybridization on a biochip carried out at stage (c). 2. A set of oligonucleotide probes, represented by the sequences of SEQ ID NO: 61-120, required to identify genetic markers of the polygenic risk of developing hormone-positive breast cancer subtype by the method according to claim 1.

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