Association of genetic polymorphism with the mutation status of the BRCA1/2 genes in spontaneous breast cancer

S. I. Polinyk, L. A. Rybchenko, S. V. Klymenko © 2017 S. I. Polinyk et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDС 618.19-006:616-001. 28:575 (083.13)

Breast cancer is the most commonly occurring cancer among women world wide [1].In Ukraine it accounts for over 20 % of all malignant tumors and is the second most frequent cause of cancer-related death [2].
Despite the fact that most cases of breast cancer are sporadic, 25 % of them are associated with hereditary inheritance factors of high penetrance genes for the development of breast cancer, and family history remains the best indicator of their individual risk [3].Among the known predisposition genes, the BRCA1 and BRCA2 mutations have the strongest influence on the susceptibility of the disease and the risk of developing breast cancer to 85 % of the mutation carriers [4,5].
Biomedicine ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell.2017.Vol.33.N 5. P 393-400 doi: http://dx.doi.org/10.7124/bc.000962 Though the effect of high penetrance gene mutations is noticeable, they account for only about 25 % of family risk and less than 5 % of the overall incidence of breast cancer, since their frequency in the general population is very low [6].As a result of the combination of several common low penetrance genes, each of which increases the risk of breast cancer, the odds ratio (OR) is 1.2-1.5 [7.8].According to the polygonal model of inheritance, a large number of low penetrance genes may have a communicative effect on the risk [9] and the disease manifestation [10,11].
Single nucleotide polymorphism (SNPs) associated with the risk of various types of cancer has been identified through Genomewide association study (GWAS).More than 22 studies have been conducted to study breast cancer in different populations, where more than 36 loci have been associated with the hereditary predisposition to this di sease [12].
Antoniou A. et al. suggested that the hereditary predisposition to breast cancer in the carriers of mutations in the BRCA1 and BRCA2 genes may be explained in a polygenic model with a large number of low penetrance alleles, each of which slightly increases the risk, but their cumulative effect becomes quite pronounced [13].The hypothesis "common disease -common variant" was formulated, according to which the hereditary predisposition to common diseases (including oncology) is caused by many genetic variants, often found in the population [14].
GWAS has identified the genetic susceptibi lity loci associated with breast cancer risk [15][16][17].Today low penetrance SNPs [18] with a weak association with the breast cancer risk compared to high penetrance mutations such as BRCA1 or BRCA2 is identified [19].Though each option with low penetrance gives only a small increase in breast cancer, a combination of individual choices can act cumulatively leading to an increased risk (some of which are listed in Table 1).Such combinations may be useful tools for the identification of women with relative risk and prevention in the population.Relations likely are situated in the range of 1.1-1.3 and 1.2-1.6 for hetero-and homozygous genotypes respectively [8,23,24].The important fact is that although mutations in the genes BRCA1, BRCA2 cause an increased risk of breast cancer, Baynes S. demonstrated that there is no association between genetic polymorphisms and mutations in the genes BRCA1, BRCA2 (separately and in combination) with the risk of breast cancer [17,25].
The aim of the work was to establish the association of genetic polymorphisms of the FGFR2, TOX3 / LOC643714 genes carriership with mutation status of the BRCA1/2 genes in Ukrainian women with breast cancer who do not have an ionizing radiation history.

Methods
Patients.The main group of patients was formed from 62 women aged 35-60 and diagnosed to have breast cancer, which was confirmed histologically.To determine the specifics of the clinical course of the disease, the history of the disease and medical records of the patients were studied; the computer database was created.The study included women without ionizing radiation and anamnesis.The studied cohort of women was divided according to their mutated status into BRCA1/2positive and negative ones.For molecular genetic stu-dies, the samples of peripheral blood were used.
DNA isolation.DNA isolation was carried out with the standard method using the NeoPrep 100 DNA Magnet kit (NeoGene, Ukraine).Also, the genomic DNA was extracted from formalin-fixed and paraffin-embedded tissues using a Quiamp DNA Mini Kit DNA kit (Quiagen, Hilden, Germany).
Allelic-specific PCR.The genotyping of the polymorphic markers rs2981582 of the gene FGFR2 and rs3803662 of the TOX3 / LOC643714 gene was performed by allelic-specific PCR with real-time detection on the LightCycler II amplifier (Roche, Switzerland) using specific primers and probes.The probes have a fluorescence modification and a gummy dye (quencher) that suppresses fluorescence until the DNA polymerase, due to its exonuclear activity, releases fluorochrome in the process of elongating the PCR product.Each step was accompanied by the registration of the fluorescence signal in the bands corresponding to fluorores fluorescence intervals.Primers for the determination of the studied polymorphisms of the FGFR2, TOX3 / LOC643714 genes synthesized by the TIB MOLBIOL company (Germany) are presented in Table 2.
The reaction mixture consisted of probes manufactured by Roche Diagnostics (Ger many).Amplification was performed under the following conditions: initial denaturation for 10 min at 95 °C; 45 amplification cycles that exponentially increase the number of ampli- recipe for growth of fibroblasts 1,26 [8,17] Breast cancer in postmeno pause [17], stronger associations with ERpositive and PR-positive breast tumors [22] no yes rs3803662 16q12.1 TOX3 DNA-depen dent regulation of the transcription 1,28 [23] 1,20 [8] Stronger associations with ER-positive breast tumors [23] yes yes At the end of the amplification reaction, the accounting and analysis of the results were made in accordance with the manufacturer's recommendations.
The search for mutations was done using the PCR method and the analysis of the melting point Tm of the amplicons obtained.The PCR reaction was performed using TIB MOLBIOL (Germany) reagents.The displacement of Tm indicated changes in the nucleic acid sequence of the amplicon.The Tm analysis was performed using Light Cycler Software (build 4.1.1.21.) (Roche, Switzerland).
Statistic.The obtained results were processed using variation statistic methods, adopted for biological research and recommended for the processing of the results of molecular genetic studies using the StatPlus Pro package.

Results
Antoniou A. C. and Easton D. F. made the hypothesis that the risk of developing breast cancer in the carriers of mutations in the BRCA1 and BRCA2 genes is modified by genetic factors [7].The multicentre studies conducted by Antoniou A.C. et al. who included in total more than 25,000 mutation carriers in the BRCA1 and BRCA2 [20,21] genes showed a modifying role of the common low penetrance genetic variants associated with the risk of development in the general population (Table 1) .
It is important that the relative risk for these genetic variants in the group of patients with mutations in the BRCA1 and BRCA2 genes coincides with the risk for the population as a whole, although it is a significant factor that modifies the risk of developing breast cancer.Thus, the data obtained most fit to a simple multiplicative interaction model, in which the effect of each variant is independent; the accurate assessment of the risk is made taking into account the contribution of high-penetrance mutations [26].Patients with mutations in the BRCA2 in postmenopause [17], stronger associations with ER-positive and PR-positive breast tumors [22].
As a result of molecular-genetic analysis of DNA in women with breast cancer without radiation history, the association of carriership of genetic polymorphisms rs2981582 of the FGFR2 gene, rs3803662 of the TOX3/ LOC643714 gene with mutation status of the genes BRCA1/2 was established.
Among the patients without breast cancer who had molecular genetic determination of genotypes of rs2981582 polymorphism of [the] FGFR2 gene, 5 of 62 subjects had positive status of the BRCA1 gene.The BRCA2 mutations were not found in the studied cohort.
In the cohort of patients without a radiation history, which managed to successfully amplify the rs3803662 polymorphism of the TOX3 / LOC643714 gene, four BRCA1positive mutations were identified among 41 patients.Instead, the BRCA2 mutation was not found in the studied group.
Having applied Fisher's ratio test we managed to calculate the association among the BRCA1 / 2 gene status indicators, depending on the variants of the genetic polymorphisms rs2981582 of the FGFR2 gene, rs3803662 of the TOX3 / LOC643714 gene.
We obtained the following results: Fisher's ratio test with the association of genetic polymorphism rs2981582 of the FGFR2 gene and rs3803662 of the TOX3 / LOC643714 gene with positive status of the BRCA1 gene was 0.212, p < 0.05 and 0.028, p > 0.05, respectively.
No association of the rs2981582 genotypes of the FGFR2 gene with positive or negative status of the BRCA1/2 genes was found in the cohort of patients with breast cancer without ionizing radiation impact in their history.
Instead, we revealed an association with the BRCA1-positive status of the minor allele in the TT polymorphism rs3803662 of the TOX3 / LOC643714 gene among patients with breast cancer without ionizing radiation in their history (Fisher's ratio test -0.028,p > 0.05).
The results of the correlation of the genetic polymorphisms rs2981582 of the FGFR2 gene and rs3803662 of the gene TOX3 / LOC643714 among patients with breast cancer without ionizing radiation in their history are pre sen ted in Table 4.
No statistic association was found among homozygotes for major alleles and heterozygote polymorphisms of the FGFR2, TOX3 / LOC643714 genes, with or without BRCA1 / 2 mutations (p < 0.05).
Thus, by comparing and analyzing the data of the molecular study, regarding the genetic polymorphism rs2981582 of the FGFR2 gene, with a positive and negative BRCA-status, no significant difference has been found between the indices.
According to the literature, the genetic polymorphism rs2981582 of the FGFR2 gene was significantly associated with the BRCA2 mutation (p = 2 × 10-8) [27].Latif et al. have also found that in the breast cancer patients who were TT genotype carriers the FGFR2 gene was associated with the positive status of the BRCA2 mutation.
In the mutation carriers, BRCA1 and BRCA2 were associated with the genetic polymorphism rs3803662 of the TOX3 / LOC643714 gene and an increased risk of breast cancer (p = 0.004 and 0.009 respectively) [28].
According to the Antoniou A. C. et al., research for the rs2981582 polymorphisms of the FGFR2 gene, an increased risk of breast cancer for BRCA2 mutation carriers was found [21].
In the study conducted in the Chilean population of women with a family history of breast cancer, a correlation was found between the minor allele rs3803662 of the TOX3/LOC643714 gene with the risk of developing breast cancer (OR = 1.5795 % CI 1.25-1.95)[29].
However, in our study, we did not have patients with such mutations.According to the same authors, the close connection between the T/T genotype and the rs3803662 polymorphism of the TOX3/LOC643714 gene with a positive mutation status of the BRCA1 gene was proved, which was reflected in our study for spontaneous breast cancer (p = 0.03).The latter fact can be a consequence of the inherited gene linkage.

Conclusions
The association of the TT rs3803662 genotype of the TOX3/LOC643714 gene with positive mutation status of the BRCA1 gene (p = 0.03) was observed in women with breast cancer without ionizing radiation in their history.
No association was found between homozygotes for major alleles and heterozygote polymorphism of the TOX3 / LOC643714 gene, and the presence or absence of the BRCA2 mutations (p < 0.05).
No statistic association was found among homozygotes for major alleles and heterozygotes of the FGFR2 polymorphism, with or without mutations in the BRCA1 and BRCA2 genes (p < 0.05).