ASSOCIATION BETWEEN THE RS801460-POLYMORPHISM IN THE SRA1 GENE AND THYROID NODULES AMONG UKRAINIAN WOMEN WITH PROLIFERATIVE TYPE OF BENIGN BREAST DYSPLASIA WITHOUT ATYPIA.

(48.6 %) and 16 (15.2 %), respectively. Significant difference was found in genotypes distribution between patients with and without nodules (P = 0.017). Multivariable logistic regression has shown that CT-genotype has lower thyroid nodules risk compared to CC- and TT-genotypes (P = 0.020, OR = 0.083, 95% CI = 0.010-0.681). Conclusion. A statistically significant association was found between SRA1 rs801460-polymorphism and thyroid nodules occurrence in Ukrainian females with proliferative type of BBD without atypia. Individuals with CT-genotype have less risk of thyroid nodules development compared to homozygotes (CC and TT).


Introduction
It was reported that only 2 % of the human genome encodes proteins. The remaining part can be represented by non-coding sequences. One of the key molecular factors of tumorigenesis is exactly noncoding RNAs. The steroid receptor RNA activator 1 (SRA1) is a long non-coding RNA that can act through the steroid hormone receptors transactivation [1, 2]. Thus, a potential role for SRA1 in the pathogenesis of hormone-sensitive tissue tumors is suggested, for example, mammary gland.
According to the World Health Organization (WHO), breast cancer (BC) is the first most common cause of cancer mortality among females in Ukraine. Therefore, it is very important to prevent the development of this pathology in time and early diagnosis is critical [3]. Due to this, pre-cancerous mammary conditions are detected, for example, benign breast dysplasia (or benign breast disease).
BBD is a heterogeneous group of lesions that includes all non-cancerous conditions of the breast [4]. It is one of the most important risk factors for BC. BBD is divided into non-proliferative disease, a proliferative disease without atypia, and proliferative disease with atypia. The presence of proliferation and atypia increases the risk of BC [5].
Luo et al. and Schonfeld et al. have found that BBD can be associated with a thyroid cancer increased risk [4,6]. Also, it has been identified that patients with history of a thyroid cancer have an increased risk of BC [7]. This may mean that a common mechanism lies in the pathogenesis of BBD and thyroid cancer. However, which ones have not yet been established. These authors studied postmenopausal women and thyroid cancer. Unlike them, we decided to check whether BBD in women, regardless of menopause, is associated with thyroid nodules. Thus, the purpose of our study is to establish whether there is an association between the SRA1 rs801460-polymorphism and the development of thyroid nodules in Ukrainian females with a proliferative type of BBD without atypia.

Study population
The whole venous blood of 117 Ukrainian women with a proliferative type of BBD without atypia (mean age [±SD] 30.43 ± 9.22) was used for the study. 12 subjects with thyroid nodules (mean age 33.92 ± 14.18) were identified among them (Table 1). Genotyping The whole venous blood of 117 subjects was used in the study. DNA extraction was performed using the GeneJET Whole Blood Genomic DNA Purification Kit (Thermo Fisher Scientific, USA). The polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) was used for the SRA1 rs801460 single nucleotide polymorphism (SNP) genotyping. The essence of SRA1 rs801460-polymorphism is the replacement of Cytosine (C) to Thymine (T) in the 140,552,345th position of chromosome 5 [8]. The following specific primers were selected: forward -5`(140552495)TTT TTA GTA GAG ACA GGG TTT TGC C(140552471)3` and reverse -5`(140552318)ACT CTA CGC CAG ACA ATA TGC TAT G(140552342)3`.
To separate the restriction products, the horizontal electrophoresis (10 V/cm) in 2.5% agarose gel with the addition of a bromide ethidium solution (10 mg/ml) was used.

Statistical analysis
The Statistical Package for Social Science software (SPSS, version 25.0, Chicago, IL, USA) was used to perform all calculations. Kolmogorov-Smirnov test confirmed the normal distribution. It was used chi-square (χ 2 ) test and two-tailed Student's t-test in the study. The logistic regression estimated the odds ratio (OR) and 95% confidence interval (CI) in the framework of additive, dominant and overdominant models of inheritance. It was used multivariable logistic regression for age and BMI adjustments. It was accepted P < 0.05 value as significant.

Results
There were 12 subjects (mean age 33.92 ± 14.18) with and 105 subjects (mean age 30.03 ± 8.48) without thyroid nodules in patients with a proliferative type of BBD without atypia. No significant differences were found in mean age as well as in BMI in comparison groups (P > 0.05). At the same time, significant difference was found in average weight (P = 0.004) and growth (P = 0.024) between these two groups (Table1).
It was revealed no significant differences in height of the glandular and fibroglandular parts of the breast (P > 0.05) ( Table 2). BBDbenign breast dysplasia; nnumber of cases; Pindicator of statistical significance; F -the ratio of the between-class scatter to the within-class scatter. Quantitative variables were compared by t-test.
The differences in the SRA1 rs801460 SNP genotype distribution in individuals with a proliferative type of BBD without atypia depending on the thyroid nodules occurrence are shown in Table 3. There were 11 estimated cases (91.7 %) of CT-genotype, 1 case (8.3 %) of CC-genotype and 0 of TT-genotype in patients with thyroid nodules.
Fifty-one subjects (48.6 %) of CT-heterozygotes, 38 subjects (36.2 %) of CC-homozygotes and 16 (15.2 %)of TT-homozygotes were revealed among patients without thyroid nodules. It was found a statistically significant differences (χ 2 = 8.110; P = 0.017). The results of the association analysis between the SRA1 rs801460-polymorphism and the thyroid nodules development in patients with a proliferative type of BBD without atypia are summarized in Table  4. Significant association between SRA1 rs801460 SNP and thyroid nodules was revealed under overdominant (P = 0.021, OR = 0.086, 95% CI = 0.011-0.689) and additive (P = 0.048, OR = 0.122, 95% CI = 0.015-0.986for CT-and CC-genotypes) models of inheritance. After adjusting for covariates of age and BMI genotypic association of rs801460 SNP remained only under over-dominant (P = 0.020, OR = 0.083, 95% CI = 0.010-0.681) model. According to this, the CT-genotype has lower thyroid nodule risk compared to CC-and TT-genotypes in patients with a proliferative type of BBD without atypia. ) BBDbenign breast dysplasia; P c : crude P value; ORc: crude odds ratio; CI: confidence interval; P a : P value adjusted for age and BMI; OR a : adjusted odds ratio

Discussion
The association between SRA1 rs801460polymorphism and the development of thyroid nodules in patients with a proliferative type of BBD without atypia was checked in this study. The SRA1 is located at reverse strand of chromosome 5 (5q31.3) and has a 687 bp core sequence [8,9]. SRA1 was discovered in 1999 by Lanz et al. [10]. They concluded that the main mechanism of its action is the coactivation of steroid hormone receptors. Therefore, a potential role of SRA1 in the pathogenesis of hormone-sensitive tissue tumors was suggested. After that, these scientists generated a transgenic-mouse model with SRA1 overexpression, whereby cell proliferation and apoptosis were found. Nevertheless, they elicited that its overexpression does not lead to tumor progression, because increased cell proliferation causes apoptosis [1].
Cooper et al. reported that SRA1 overexpression has a statistically significant association with breast tumors with higher progesterone receptor contents. In contrast to Lanz et al., they found that the SRA1 may be involved in tumorigenesis and breast cancer progression [11].
Among all the SRA1 polymorphisms, we chose rs801480, the essence of which is the C to T replacement in the 140,552,345th position of the (+)chain of chromosome 5 [8]. Yan et al. studied the association between SRA1 rs10463297 and rs801460 SNPs and breast cancer development. They identified rs801460 GA-, AA-, and GA-+ AAgenotypes, as well, as rs10463297 TC-and TC-+ CC-genotypes associated with estrogen receptor (ER) positivity in BC patients [2].
Interestingly, tumor processes in mammary and thyroid glands are associated. Luo et al. investigated the possible association between BBD and thyroid cancer development. They established that postmenopausal females with BBD had an increased risk of thyroid cancer than females without BBD [4]. The same conclusion was made by Schonfeld et al. [6]. Patients with a thyroid cancer history also can have an increased risk of BC. Thyroid cancer survivors were found by Kuo et al. to have a higher risk for breast cancer development than the general population [7].

Conclusions
1. There is an association between SRA1 rs801460-polymorphism and the development of thyroid nodules in Ukrainian females with proliferative type of BBD without atypia.