RU2828525C1 - Method for prediction of risk of developing breast cancer in women without obesity by results of genetic testing - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to clinical oncology, medical genetics and molecular diagnostics, and can be used to predict the risk of breast cancer in women without obesity of Russian nationality, which are natives of the Central Black Earth Region of the Russian Federation. DNA is recovered from peripheral venous blood. Carried out is analysis of polymorphic markers rs8023580 of the NR2F2 gene, rs4149056 of the SLCO1B1 gene and rs440837 of the ZBTB10 gene. When detecting a combination of genotypes rs8023580-TT of the NR2F2 gene – rs4149056-TT of the SLCO1B1 gene – rs440837-GG of the ZBTB10 gene high risk of developing breast cancer in women without obesity is predicted.

EFFECT: method provides obtaining new criteria for assessing an increased risk of developing breast cancer in women without obesity of Russian nationality, natives of the Central Black Earth Region of the Russian Federation, based on data on polymorphic markers rs8023580 NR2F2, rs4149056 SLCO1B1 and rs440837 ZBTB10.

1 cl, 3 dwg, 5 ex

Description

The invention relates to the field of medicine, in particular to clinical oncology, medical genetics, molecular diagnostics, and can be used to predict the risk of developing breast cancer in women without obesity based on the results of genetic testing.

Breast cancer is the most common oncological disease in women worldwide [WHO Information Materials, 2023. https://www.who.int/ru/news-room/fact-sheets/detail/breast-cancer]. Today, there is a wealth of data showing the influence of lifestyle and environmental factors on the development of breast cancer. Such factors include a high-fat diet, alcohol consumption, and insufficient physical activity. These factors can be corrected, which entails a decrease in morbidity and mortality [A.B. Shertaeva, D.A. Ospanova, A.M. Grzhibovsky, A.S. Aubakirova, T.R. Fazylov. Risk factors for breast cancer / Bulletin of KazNMU No. 1.-2022. P. 192-202].

It is known that genetic and environmental factors, acting together, significantly increase the risk of breast cancer. Environmental and behavioral factors often include: ionizing radiation, hormonal therapy, reproductive behavior (eg, late age at first birth) [Powell M, Jamshidian F, Cheyne K, Nititham J, Prebil LA, Ereman R. Assessing breast cancer risk models in Marin County, a population with high rates of delayed childbirth. Clin Breast Cancer. 2014;14(3):212-220.e1. doi:10.1016/j.clbc.2013.11.003], alcohol, dietary factors, obesity, and insufficient physical activity [Coughlin SS, Smith SA. The Impact of the Natural, Social, Built, and Policy Environments on Breast Cancer. J Environ Health Sci. 2015;1(3):10.15436/2378-6841.15.020. doi:10.15436/2378-6841.15.020]. Excessive consumption of high-calorie foods leads to weight gain and, ultimately, obesity, which is associated with an increased risk of cancer [Davoodi SH, Malek-Shahabi T, Malekshahi-Moghadam A, Shahbazi R, Esmaeili S. Obesity as an important risk factor for certain types of cancer. Iran J Cancer Prev. 2013;6(4):186-194].

Obesity, defined today as a body mass index (BMI) >30 kg / m², affects more than 13% of the adult population of the planet, which is more than 600 million people [World Health Organization. Obesity and overweight. World Health Organization, 2017. Available at: https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight]. Overweight and obesity are associated with an increased risk of developing cancer of the esophagus, stomach, thyroid, colon, pancreas, rectum, uterus, prostate, gallbladder, ovaries, and breast [World Cancer Research Fund (WCRF). Continuous Update Project: 2016. London: WCRF International, 2016.]. An increase in BMI by 5 kg / m2 increases the risk of developing breast cancer by 12% [Renehan A.G., Tyson M., Egger M. et al. Body-mass index and incidence of cancer: A systematic review and meta-analysis of prospective observational studies. Lancet 2008;371:569–78]. Overweight women with breast cancer have worse cancer-specific survival outcomes [Protani M., Coory M., Martin J.H. Effect of obesity on survival of women with breast cancer: Systematic review and meta-analysis. Breast Cancer Res Treat 2010;123:627–35.]. Obesity is associated with an increased risk of death from breast cancer in both pre- and postmenopausal women [Chan D.S., Vieira A.R., Aune D. et al. Body mass index and survival in women with breast cancer – systematic literature review and meta-analysis of 82 follow-up studies. Ann Oncol 2014;25:1901–14].

Breast cancer has been actively studied from a genetic standpoint (twin, family, association, genome-wide (GWAS) studies) in recent decades. Considerable factual material has been accumulated on this issue, convincingly demonstrating the significant contribution of hereditary factors to susceptibility to the disease. According to data from large-scale twin studies performed in European populations and including materials on several hundred [Mucci LA, Hjelmborg JB, Harris JR, et al. Familial Risk and Heritability of Cancer Among Twins in Nordic Countries [published correction appears in JAMA. 2016 Feb 23;315(8):822]. JAMA. 2016;315(1):68-76. doi:10.1001/jama.2015.17703] thousand pairs of twins, the contribution of “genetics” to the development of breast cancer is 31%.

In the Russian Federation, studies of the involvement of polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene in the formation of predisposition to breast cancer in women without obesity based on the results of genetic testing are rare and fragmentary, and data on the role of polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene in the development of breast cancer in women without obesity based on the results of genetic testing are absent.

To assess the current patent situation, a search was conducted on security documents for the period from 1990 to 2023. The documents were analyzed in the following direction: a method for predicting the risk of developing breast cancer in women without obesity based on the results of genetic testing. Sources of information: websites of the Federal Institute of Industrial Property http://fips.ru.

In the reviewed scientific, medical and available patent literature, the authors did not find a method for predicting an increased risk of developing breast cancer in women without obesity based on data on polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene.

Known is RU patent No. 2642623 (published 25.01.2018), which describes a method for predicting the risk of developing a malignant neoplasm or diagnosing a malignant neoplasm in a female. The invention relates to diagnostics, in particular a method for predicting the risk of developing breast cancer in a female who does not suffer from breast cancer. The method for predicting the risk of developing breast cancer in a female who does not suffer from breast cancer includes: determining the level of pro-neurotensin 1-117 with the amino acid sequence presented in SEQ ID No. 5, or its fragments of at least 5 amino acids, or peptides containing this pro-neurotensin 1-117, in a biological fluid obtained from the said female individual, selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid and saliva, where the level of pro-neurotensin 1-117 is determined on an empty stomach; and a correlation of the said level of pro-neurotensin 1-117 or its fragments or peptides containing pro-neurotensin 1-117 with the risk of developing breast cancer, where an elevated level is prognostic for an increased risk of developing breast cancer. The above-described method allows for effective prediction of the risk of developing breast cancer in a female individual. The disadvantages of this technical solution are the labor intensity of the method for its implementation, invasiveness and potential trauma, especially when collecting cerebrospinal fluid during a spinal puncture.

Known patent RU No. 2717148 (published 18.03.2020), which describes a method for selecting women in a risk group for developing breast cancer in the perimenopausal period. The invention relates to medicine, namely to oncology, and can be used to select women in a risk group for developing breast cancer in the perimenopausal period. Anamnestic data is collected. Women aged 44 to 58 years with at least two births and no more than 1 abortion in their history are identified, the age of the first pregnancy and the sex of the children born are determined. If the first birth occurred at the age of 23 and resulted in the birth of a male child, and the second birth resulted in the birth of a female child, the level of estrogen metabolites 2-hydroxyestrone (2-ONE1) and 16α-hydroxyestrone (16α-ONE1) is additionally determined in daily urine, and their ratio 2-ONE1/16α-ONE1 is calculated. If the 2-ONE1/16α-ONE1 index value of 1.64 or less is detected, then this woman is classified as being at high risk for developing breast cancer. The method provides a simple express method for determining the risk of developing breast cancer in women in the perimenopausal period due to the analysis of anamnestic data and determination of the level of estrogen metabolites 2-ONE1 and 16α-ONE1 in daily urine. The disadvantages of this technical solution are the invasiveness of the method and the limited time frame for its use (the method is effective only during the menopausal period), and genetic data are not taken into account.

Known is RU patent No. 2671578 (published 02.11.2018), which describes a method for predicting the risk of cancer or diagnosing cancer in a woman. The invention relates to medicine and concerns a method for predicting the risk of breast cancer or lung cancer in a woman who does not suffer from cancer, including determining the level of proenkephalin or its fragments consisting of at least 5 amino acids in total body water taken from said woman, and establishing a correlation between said level of proenkephalin (PENK) or its fragments and the risk of breast cancer or lung cancer. In this case, a reduced level is a prognostic criterion for an increased risk of breast cancer or lung cancer. The invention provides an effective method for predicting the risk of breast cancer or lung cancer in a woman who does not suffer from cancer. The disadvantages of this technical solution are the labor intensity and high cost of the method, which consists of the need to use special biochemical kits, which are expensive and inaccessible to outpatient clinics.

Known is the patent RU No. 2480763 (published 27.04.2013), which describes a method for predicting the risk of developing benign breast dysplasia in women with genital endometriosis. The present invention relates to the field of medicine, in particular to molecular genetic studies, and describes a method for predicting the risk of developing benign breast dysplasia in women with genital endometriosis, including the extraction of DNA from peripheral venous blood, determination of the polymorphism of the gene of the receptor of the tumor necrosis factor α type 1 (+36 A/G TNFR1), wherein upon detection of the genotypes +36 AA TNFR1 and +36 AG TNFR1, a high risk of developing benign breast dysplasia in genital endometriosis is predicted. The use of the method allows predicting the risk of benign hyperplasia of the mammary gland among patients with genital endometriosis and, based on this, determining further tactics for managing patients with genital endometriosis. The disadvantage of the known method is that it includes data on only one polymorphic locus.

Patent RU No. 2522501 (published 20.07.2014) describes a method for predicting hereditary predisposition to breast cancer. The invention relates to the field of medicine, in particular, to clinical oncology, medical genetics, molecular diagnostics and can be used to predict hereditary predisposition to breast cancer. The method is characterized in that short fragments of the BLM gene up to 200 bp in length are amplified, followed by high-resolution melting, including a heteroduplex formation step optimized for the BLM gene: rapid heating to 95°C and slow temperature reduction to 50°C; one fragment with an aberrant melting profile is selected for sequencing, the selected fragment is sequenced and, if a mutation in the BLM gene is detected, hereditary predisposition to breast cancer is predicted. The method increases the accuracy of mutation detection, is easy to perform and highly informative: it allows to detect up to 100% of mutations in the BLM gene. The disadvantage of this method is its labor intensity, it does not take into account the role of genetic polymorphisms.

Known patent RU No. 2795726 (published 11.05.2023), which describes a method for predicting the risk of developing breast cancer in women without obesity. The method includes: isolating DNA from peripheral venous blood, analyzing genetic polymorphisms rs3918242-rs3918249-rs17576 of the MMP-9 gene, which are prognostically significant factors in the development of breast cancer. The CCA haplotype of the polymorphic loci rs3918242-rs3918249-rs17576 of the MMP-9 gene is a risk factor for developing breast cancer in women without obesity.

The objective of this study is to expand the range of diagnostic methods, namely, to create a method for predicting the risk of developing breast cancer in women without obesity based on data on polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene.

The technical result of using the invention is obtaining criteria for assessing the increased risk of developing breast cancer in women without obesity of Russian nationality, natives of the Central Black Earth Region of the Russian Federation, based on data on polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene, including:

- DNA extraction from peripheral venous blood;

- analysis of polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene;

- prediction of the risk of developing breast cancer in women without obesity when identifying a combination of genotypes rs8023580-TT of the NR2F2 gene - rs4149056-TT of the SLCO1B1 gene - rs440837-GG of the ZBTB10 gene.

The novelty and inventive step consist in the fact that the prior art does not disclose the possibility of predicting the risk of developing breast cancer in women without obesity based on data on the combination of genotypes rs8023580-TT of the NR2F2 gene - rs4149056-TT of the SLCO1B1 gene - rs440837-GG of the ZBTB10 gene.

The method is carried out as follows:

Isolation of genomic DNA from peripheral blood is carried out by the phenol-chloroform extraction method (Miller, S. A. A simple salting out procedure for extracting DNA from human nucleated cells / S. A. Miller, D. D. Dykes, H. F. Polesky // Nucleic. Acids. Res. - 1988. - Vol. 16, No. 3. - P. 1215) in two stages. In the first stage, 25 ml of lysis buffer containing 320 mM sucrose, 1% Triton X-100, 5 mM MgCl ₂ , 10 mM Tris-HCl (pH = 7.6) are added to 4 ml of blood with EDTA. The resulting mixture is mixed and centrifuged at 4 °C, 4000 rpm for 20 minutes. After centrifugation, the supernatant is poured off, 4 ml of a solution containing 25 mM EDTA (pH = 8.0) and 75 mM NaCl are added to the sediment, and the mixture is resuspended. Then 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg/ml) are added, and the sample is incubated at 37ºC for 16 hours.

In the second stage, DNA is extracted from the obtained lysate sequentially with equal volumes of phenol, phenol-chloroform (1:1), and chloroform with centrifugation at 4000 rpm for 10 minutes. After each centrifugation, the aqueous phase is collected. DNA is precipitated from the solution with two volumes of cooled 96% ethanol. After lyophilization, the obtained DNA is dissolved in bidistilled, deionized water and stored at -20 ⁰ C.

Analysis of polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene was carried out by the polymerase chain reaction (PCR) method on a CFX-96 Real-Time System thermal cycler (Bio-Rad) using standard oligonucleotide primers and probes (synthesized at Test-Gen LLC (Ulyanovsk)).

Amplification of genomic DNA was performed in a reaction mixture with a total volume of 10 μl, including a mixture for PCR of rs8023580 of the NR2F2 gene or rs4149056 of the SLCO1B1 gene or rs440837 of the ZBTB10 gene – 4 μl, Taq polymerase - 2 μl, the test sample (~30 ng DNA/μl) - 1 μl, deionized water – 3 μl.

Genotyping of the studied samples was carried out using the CFX-Manager™ software using the allele discrimination method based on the relative fluorescence units (RFU) values (Fig. 1, Fig. 2, Fig. 3).

The invention is characterized by the following figures:

Fig. 1 – Visualization of discrimination of rs8023580 NR2F2 genotypes (where - TT homozygotes, - TC heterozygotes, - CC homozygotes, - unspecified sample).

Fig. 2 – Visualization of discrimination of rs4149056 SLCO1B1 genotypes (where - TT homozygotes, - TC heterozygotes, - CC homozygotes, - undefined sample)

Fig. 3. – Visualization of discrimination of rs440837 ZBTB10 genotypes (where - homozygotes AA, - heterozygotes AG, - homozygotes GG, - unspecified sample).

At the next stage of the work, interlocus interactions that have risk values for breast cancer were modeled in the MB-MDR software package (access-https://github.com/imbs-hl/mbmdR) (covariates were taken into account and permutation procedures were performed). Models with the participation of 2-, 3-, 4-, 5- loci were estimated. In this case, in order to reduce the probability of false positive results for “selecting” the most significant models that determine the risk of developing the disease (at each level we selected 2-3 most significant models), at the stage of selecting models for permutation testing, we additionally introduced the Bonferroni correction taking into account possible combinations of the 9 loci under consideration: for 2 locus models, the “threshold” level was set at p=0.05/36 = 1.39*10-3, for 3 locus models - p=0.05/84 = 5.95*10-4, for 4 locus models - p=0.05/126 = 3.97*10-4, for 5 locus models - p=0.05/126 = 3.97*10-4. Visualization of interlocus interactions associated with breast cancer was performed using the MDR program (available at http://www. multifactordimensionalityreduction.org/).

The possibility of using the proposed method for assessing the risk of breast cancer development in women without obesity is confirmed by the analysis of the results of observations of 1126 non-obese patients, including 239 patients and 887 patients in the control group. The age characteristics of the patients and controls were comparable. The study samples included: 1) patients of Russian nationality, natives of the Central Black Earth Region of the Russian Federation, unrelated to each other and living in the Belgorod Region (Churnosov M.I., Sorokina I.N., Balanovskaya E.V. Gene pool of the population of the Belgorod Region. Dynamics of the endogamy index in district populations // Genetics. 2008. Vol. 44. No. 8. pp. 1117-1125), who voluntarily agreed to the study; 2) patients were included in the group of patients only after a diagnosis of breast cancer was established, confirmed using clinical and laboratory-instrumental (including morphological) examination methods.

The examination of patients with breast cancer was carried out at the outpatient and chemotherapy departments of the Belgorod Regional Oncology Dispensary; the formation of a control group (without clinical and anamnestic signs of breast cancer) was carried out at the perinatal center of the Belgorod Regional Clinical Hospital of St. Joasaph (during professional examinations).

All breast cancer patients and women in the control group signed informed consent to participate in the study (the study was approved by the Ethics Committee of the Medical Institute of the National Research University "BelSU").

Typing of molecular genetic markers was carried out at the Department of Medical and Biological Disciplines of the Medical Institute of the National Research University "BelSU".

When studying SNP x SNP interactions, a genetic model was established that includes the presence of a significant three-locus model involved in the formation of breast cancer in women without obesity, which is rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene (p _perm ≤0.001). With the development of the disease, the most significant association was found for the combination of genotypes rs8023580-TT of the NR2F2 gene - rs4149056-TT of the SLCO1B1 gene - rs440837-GG of the ZBTB10 gene (beta = 1.154; p = 0.023), which has a risk direction.

As examples of the specific application of the developed method, a genetic examination of women of Russian nationality, who are natives of the Central Black Earth Region of the Russian Federation and are not related to each other, was conducted: a genetic study was conducted on the polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene.

Venous blood was taken from patient C., genotyping of DNA markers was performed, during the analysis of the involvement of polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene, a combination of genotypes rs8023580-TT of the NR2F2 gene - rs4149056-TT of the SLCO1B1 gene - rs440837-GG of the ZBTB10 gene was detected, which allowed us to classify the patient into the group of patients with an increased risk of developing breast cancer in women without obesity. Further observation confirmed the diagnosis of breast cancer in the patient.

Venous blood was taken from patient B., genotyping of DNA markers was performed, during the analysis of the involvement of polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene, a combination of genotypes rs8023580-TC of the NR2F2 gene - rs4149056-TC of the SLCO1B1 gene - rs440837-AG of the ZBTB10 gene was identified, which allowed us to classify the patient into the group of patients with a low risk of developing breast cancer in women without obesity. Further observation did not confirm the diagnosis in the patient.

Venous blood was taken from patient Ya., genotyping of DNA markers was performed, during the analysis of the involvement of polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene, a combination of genotypes rs8023580-CC of the NR2F2 gene - rs4149056-TC of the SLCO1B1 gene - rs440837-AG of the ZBTB10 gene was revealed, which allowed us to classify the patient into the group of patients with a low risk of developing breast cancer in women without obesity. Further observation did not confirm the diagnosis of breast cancer in the patient.

Venous blood was taken from patient H., genotyping of DNA markers was performed, during the analysis of the involvement of polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene, a combination of genotypes rs8023580-TT of the NR2F2 gene - rs4149056-TT of the SLCO1B1 gene - rs440837-GG of the ZBTB10 gene was identified, which allowed us to classify the patient into the group of patients with an increased risk of developing breast cancer in women without obesity. Further observation confirmed the diagnosis of breast cancer in patient H.

Venous blood was taken from patient I., genotyping of DNA markers was performed, during the analysis of the involvement of polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene, a combination of genotypes rs8023580-CC of the NR2F2 gene - rs4149056-CC of the SLCO1B1 gene - rs440837-AA of the ZBTB10 gene was detected, which allowed us to classify the patient into the group of individuals with a low risk of developing breast cancer in women without obesity. Upon further observation, the diagnosis of breast cancer in patient I. was not confirmed.

The use of this method will allow the formation of risk groups among women at the preclinical stage and the timely implementation of the necessary treatment and preventive measures in these groups to prevent the development of breast cancer in women without obesity.

Claims (1)

A method for predicting the risk of developing breast cancer in women without obesity of Russian nationality, who are natives of the Central Black Earth Region of the Russian Federation, including the extraction of DNA from peripheral venous blood, analysis of polymorphic markers rs8023580 of the NR2F2 gene - rs4149056 of the SLCO1B1 gene - rs440837 of the ZBTB10 gene, predict a high risk of developing breast cancer in women without obesity when identifying a combination of genotypes rs8023580-TT of the NR2F2 gene - rs4149056-TT of the SLCO1B1 gene - rs440837-GG of the ZBTB10 gene.