Positive association between SRA1 rs801460 variant and proliferative type of benign breast disease with atypia in Ukrainian females
Summary. Aim: To investigate the association between SRA1 rs801460 and rs10463297 variants and proliferative type of benign breast disease with atypia development in Ukrainian females. Materials and Methods: 83 individuals diagnosed with proliferative type of benign breast disease with atypia and 115 without atypia were enrolled in the study. The rs801460 and rs10463297 variants genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism analysis. Hematoxylin and eosin, toluidine blue and van Gieson’s picrofuchsin methods were used for sections staining. Results: It was revealed that SRA1 rs801460-variant is associated with proliferative type of benign breast disease with atypia development both before and after adjustment for risk factors (age, body mass index, age of menarche, oral contraceptives intake and burdened history of breast cancer). The risk for mentioned disease in the individuals with rs801460 TT-genotype is 2.2 times higher (confidence interval 1.010–4.800; p = 0.047) than in individuals with the CC and CT genotypes. No link between SRA1 rs10463297 and proliferative type of benign breast disease with atypia occurrence in Ukrainian females was found. Conclusion: The present study specified that SRA1 rs801460, but not rs10463297, can be the strong genetic predictor for benign breast disease with atypia in Ukrainian females.
Submitted: February 9, 2021.
*Correspondence: E-mail: email@example.com
Abbreviations used: ANDI — aberrations of normal development and involution; BBD — benign breast disease; BC — breast cancer; BMI — body mass index; CI — confidence interval; ER — estrogen receptor; OR — odds ratio; PCR-RFLP — polymerase chain reaction-restriction fragment length polymorphism analysis; SNV — single nucleotide variant; SRA — steroid receptor RNA activator.
Breast cancer (BC) is the most commonly occurring cancer in women and the second most common cancer overall . Many BC survivors need rehabilitation because of physical, psychological and social dysfunctions . Consequently, it is essential to improve early diagnosis focusing on detection of pre-malignant lesions of the breast.
Benign breast disease (BBD) is a group of benign mammary conditions, some of which can lead to the BC development. It includes nonproliferative lesions, proliferative lesions without atypia, and proliferative lesions with atypia . The latter are of the highest BC risk. Based on the aberrations of normal development and involution (ANDI) concept, the BBD origin is related to the normal breast physiology encompassing the entire spectrum of benign conditions from mild to severe abnormality [4, 5].
It is considered that estrogen may contribute to the BC development, for instance, because of enhancing breast cell proliferation. The effects of estrogen are mediated via two estrogen receptor (ER) subtypes, ERα and ERβ . These nuclear receptors expressed in the mammary tissue are capable to modulate the transcription activity of target genes [7, 8]. Only a part of breast cells contains ER while the rest are receptor-negative. Clarke et al.  showed that proliferating cells are receptor-negative and located near the receptor-positive cells. Accordingly, estrogen can modulate receptor-positive cells activity that stimulates proliferation through the paracrine signaling.
The steroid receptor RNA activator (SRA) is the long non-coding RNA (lncRNA) that also can act via the ERα- and ERβ-receptors. It is a coactivator of steroid receptors, such as ER, androgen receptor, glucocorticoid receptor (GR) and progesterone receptor. This lncRNA is encoded by the SRA1 gene, which is 8027 bp in length and located on 5q31.3 chromosome. It consists of 5 exons and 4 introns (NC_000005.10) . Lanz et al. [11, 12] found that transgenic mice with human SRA-overexpression exert an abnormal breast development. Histopathology showed ductal ectasia, ductal-epithelial hyperplasia, focal metaplasia and intraductal proliferation. Therefore, SRA may be associated with the proliferative type of BBD development.
In our previous article, we found SRA1 rs801460-variant to be associated with the development of thyroid nodules in Ukrainian females with proliferative type of BBD without atypia . The aim of the present research was to explore the possible link between SRA1 rs801460 and rs10463297 single nucleotide variants (SNVs) and proliferative type of BBD with atypia development in the Ukrainian females.
MATERIALS AND METHODS
Study population. Venous blood of 83 females with proliferative type of BBD with atypia (mean age [±SD] 36.80 ± 8.87) and 115 females with proliferative type of BBD without atypia (mean age 30.43 ± 9.26) as a control was used for the study. The licensed surgeon examined each BBD patient on an outpatient basis. Surgical treatment was performed for all patients at the clinical sites of the Department of Surgery with a Course of Pediatric Surgery and Urology of the Sumy Regional Oncology Center. The Scientific center of pathomorphological researches of the Sumy State University studied each morphological material. Molecular genetic research was conducted in the Scientific Laboratory of Molecular Genetic Research of the Sumy State University. Study involved individuals with atypical proliferative changes in mammary gland and excluded patients with nonproliferative lesions.
Patient assessment, examination and diagnosis were carried out in accordance with the current orders of the Ministry of Health of Ukraine № 690 from 23.09.2009 and № 616 from 03.08.2012, the European Convention of Human Rights and Biomedicine and the Declaration of Helsinki of the World Medical Association on Ethical Principles for Medical Research Involving Human Subjects. All participants provided a written informed consent.
Genotyping of SNVs. Genomic DNA was extracted from the whole venous blood using the GeneJET Whole Blood Genomic DNA Purification Kit (Thermo Fisher Scientific, USA). Genotyping of the SRA1 rs801460 and rs10463297 SNVs was performed using the polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP). The reaction mixture for PCR included 5 μL of FastDigest Green Buffer (10X) (Thermo Fisher Scientific, USA), 0.5 μL dNTP Mix (10 mM of each deoxynucleotide) (Thermo Fisher Scientific, USA), 0.75 U DreamTaq DNA Polymerase (5 U/μL) (Thermo Fisher Scientific, USA), 0.1 μL of each primer, 75–100 ng DNA, and bidistilled water added up to 25 μL. The sequences of the specific primers (Yan et al. ) and PCR conditions are shown in Table 1. Amplification was carried out using Thermocycler GeneAmp PCR System 2700 (Thermo Fisher Scientific, USA).
Table 1. PCR conditions for SRA1 rs801460 and rs10463297 genotyping
Notes: D — denaturation; H — hybridization; E — elongation; F — forward primer; R — reverse primer; bp — base pairs.
The reaction mixture on the restriction stage consisted of 2U of restriction enzyme, 0.8 μL of 10X Buffer R (Thermo Fisher Scientific, USA) and bidistilled water added up to 2 μL. The amplification product (6 μL) with the addition of the reaction mixture (2 μL) was incubated at 37 °C for 20 h. NsiI (Thermo Fisher Scientific, USA) was used for restriction analysis of SRA1 rs801460 SNV. The replacement of cytosine (C) to thymine (T) at the (-)5749th position of the SRA1 gene leads to the splitting of the amplicon (178 bp) by NsiI into 155 bp and 23 bp fragments. We had only one 178 bp fragment in the presence of cytosine due to the NsiI restriction site loss (Fig. 1). Restriction analysis of the SRA1 rs10463297-variant was done using Eco47I (Thermo Fisher Scientific, USA). Eco47I cut the primary amplicon (483bp) into two fragments of 317bp and 166bp in case of thymine to cytosine substitution at the (-)1440th position of the SRA1 gene. The presence of thymine prevented restriction and the primary amplicon was retained (Fig. 2).
Fig. 1. Results of SRA1 rs801460 variant restriction analysis. M — molecular marker (bp — base pairs); lanes 1 and 4 — CC-genotype; lanes 3, 6 and 7 — TC-genotype; lanes 2, 5 and 8 — TT-genotype
Fig. 2. Results of SRA1 rs10463297 variant restriction analysis. M — molecular marker (bp — base pairs); lanes 2, 4 and 5 — TT-genotype; lanes 1, 3, 7 and 8 — TC-genotype; lane 6 — CC-genotype
Restriction fragments were separated using horizontal electrophoresis (10 V/cm) with the ethidium bromide (10 mg/mL) addition in 2.5% agarose gel. Ultraviolet transillumination was applied for DNA fragments visualization.
Histology. Breast tissue obtained during surgery was used for histology (Fig. 3). Biological material was fixed in 10% phosphate buffered formalin for 48 h and then embedded in paraffin. Paraffin series were sliced at a thickness of 8–10 μm and incubated at 37 °C for 12 h. After deparaffinization, sections were stained with one of the following methods: hematoxylin and eosin, toluidine blue or van Gieson’s picrofuchsin.
Fig. 3. Simple (1) and atypical (2) intraductal papillomas. H&E, ×200 (left section), ×400 (right section)
Statistical analysis. To analyze the possible link between SRA1 rs801460 and rs10463297 SNVs and proliferative type of BBD with atypia development, informative samples were selected (198 cases). The statistical analysis was performed using Statistical Package for the Social Sciences software (SPSS, version 25.0, Chicago, IL, USA). Continuous variables are presented as the mean ± SD (Kolmogorov–Smirnov test was applied for the distribution normality checking); categorical variables are indicated as absolute number and percentage value. The mean values were compared using two-tailed Student’s t-test for two and ANOVA for three groups. Bonferroni post hoc test was used for multiple comparisons. Chi square (χ2) test was used for the comparison of alleles and genotypes frequencies as well as other categorical variables. An odds ratio (OR) and 95% confidence interval (CI) were received from logistic regression for the four models of inheritance: dominant, recessive, overdominant and additive. Multivariable logistic regression was used to increase reliability of the obtained results via adjustment for age, body mass index (BMI), age of menarche, oral contraceptives intake and burdened history of BC. Value p < 0.05 was considered significant.
The clinical characteristics of the comparison groups are presented in Table 2. The differences in age (p < 0.001) and BMI (p = 0.016) were found to be significant. In contrast, the investigated groups were comparable by height (p = 0.296), size of the glandular and fibroglandular parts of breast (p = 0.756 and p = 0.364 respectively), age of menarche (p = 0.273), oral contraceptives intake (p = 0.733) and burdened history of BC (p = 0.136).
Table 2. Clinical characteristics of the study population
Notes: Categorical variables were compared by χ2 test, continuous variables by t-test.
The distribution of SRA1 rs801460 and rs10463297 alleles and genotypes in comparison groups is indicated in Table 3. The frequency of SRA1 rs801460 genotypes in patients with proliferative lesions with atypia significantly differed from the group without atypia (p = 0.032), while the distribution of SRA1 rs10463297 genotypes was similar between these groups (p = 0.852). No significant differences between alleles were found in comparison groups for both SNVs (p > 0.05).
Table 3. Distribution of genotypes and alleles in comparison groups
Notes: Categorical variables were compared by χ2 test.
The results of SRA1 rs801460 and rs10463297 genotypic association with proliferative type of BBD with atypia are shown in Table 4. The statistically significant association was found for SRA1 rs801460-variant in a crude recessive model (Pc = 0.012; ORc = 2.602, 95% CI = 1.239–5.462) of inheritance as well as after adjustment for age, body mass index, age of menarche, oral contraceptives intake and burdened history of BC (Pa = 0.047; ORa = 2.202, 95% CI = 1.010–4.800). Although mean age of the group with atypia is markedly higher than that of the group without atypia (p < 0.001), a significant link remained after adjustment. It supports the genotypic association between SRA1 rs801460-variant and proliferative type of BBD with atypia development.
Table 4. Analysis of SRA1 rs801460 and rs10463297 genotypic association with proliferative type of BBD with atypia development
Notes: Pc — crude P value; ORc — crude odds ratio; Pa — P value adjusted for age, body mass index, age of menarche, oral contraceptives intake and burdened history of BC; ORa — adjusted odds ratio.
Estrogen is a steroid hormone that not only controls the development and normal functioning of the mammary gland, but also is involved in the breast tumorigenesis. Lanz et al.  in 1999 discovered lncRNA SRA as a ‘novel transcriptional coactivator’. It is intergenic with the core region length of 687 bp and has 3 human isoforms [8, 11]. The SRA1, steroid receptor RNA activator 1 gene, is located on 5q31.3 chromosome. It is 8027 bp in length, includes 5 exons and 4 introns (NC_000005.10)  and encodes a protein, called steroid receptor RNA activator protein .
SRA was found to be up-regulated in breast, uterus and ovary. lncRNA SRA is a coactivator of steroid receptors (ER, androgen receptor, progesterone receptor, glucocorticoid receptor), as well as other types of nuclear receptors and transcription factors . It can coactivate the hormone-independent activation domain AF-1 of ERα and ligand-dependent activation domain AF-2 of ERα and ERβ. To mediate full activation, SRA needs the presence of serine-118 in the AF-1. E2 (17-beta-estradiol) induces serine-118 residue phosphorylation by itself or indirectly through the mitogen-activated protein kinase pathway activation that is necessary for SRA functioning [15, 16]. Activated ER receptors stimulate ER-negative cells proliferation through the paracrine signaling .
It can be assumed that one nucleotide substitution may lead to a change in the level of SRA1 expression that affects cell proliferation and predisposes to BBD development. Unfortunately, there are no studies devoted to the association analysis between SRA1 variants and BBD development. The only known study is that by Yan et al.  who found that rs10463297 TC-genotype is associated with an increased BC risk in comparison with CC-genotype. In contrast, logistic regression analysis for SRA1 rs801460 did not show any significant link with BC development. In addition, both SNVs were significantly associated with ER positivity status.
BBD includes premalignant lesions that can lead to the BC development . Therefore, it is interesting to determine the influence of SRA1 rs801460 and rs10463297 variants on the development of proliferative lesions with atypia.
The essence of rs801460-variant is the replacement of cytosine by thymine at the (-)5749th position of the SRA1 gene . According to 1000 Genomes Project phase 3 browser , the minor T allele frequency is 0.43 in general population, 0.48 in European, 0.51 in Ad Mixed American, 0.22 in African, 0.56 in East Asian and 0.48 in South Asian. The polymorphic site rs10463297 is located in the SRA1 second intron as well, as rs801460 SNV, and leads to thymine/cytosine conversion at (-)1440th position. The minor C allele frequency is 0.33 in general population, 0.40 in European, 0.42 in Ad Mixed American, 0.01 in African, 0.51 in East Asian and 0.43 in South Asian .
The obtained results on the link of SRA1 rs801460 and rs10463297 variants with proliferative type of BBD with atypia showed that only the rs801460 locus is associated with the occurrence of the mentioned disease in Ukrainian females. Regardless of the adjustment for risk factors, it was found that the risk of proliferative lesions with atypia development in subjects with TT-genotype is 2.2 times higher than in individuals with CC and CT genotypes. This is the first evidence of the association between SRA1 gene variants and BBD occurrence in Ukrainian females. The comparatively small number of patients enrolled into the present study is a significant limitation. Hence, future case-control studies involving more patients are essential.
1. World Health Organization. WHO Cancer Today, 2020. Retrieved from https://gco.iarc.fr/today/home
ПОЗИТИВНИЙ ЗВ’ЯЗОК МІЖ SRA1 RS801460-ВАРІАНТОМ ТА ПРОЛІФЕРАТИВНИМ ТИПОМ ДОБРОЯКІСНОЇ ДИСПЛАЗІЇ МОЛОЧНОЇ ЗАЛОЗИ З АТИПІЄЮ СЕРЕД УКРАЇНСЬКИХ ЖІНОК
Сумський державний університет, Суми 40007, Україна
Резюме. Мета: Дослідити зв’язок між SRA1 rs801460- та rs10463297-варіантами та виникненням проліферативного типу доброякісної дисплазії молочної залози з атипією серед українських жінок. Матеріали та методи: У цьому проєкті проводилося дослідження 83 пацієнток з проліферативним типом доброякісної дисплазії молочної залози з атипією та 115 — без атипії. Метод полімеразної ланцюгової реакції з аналізом довжини рестрикційних фрагментів (PCR-RFLP) було використано для генотипування rs801460- та rs10463297-варіантів. Гематоксилін та еозин, толуїдиновий синій та пікрофуксин за Ван Гізоном використовували для фарбування зразків. Результати: У ході дослідження було виявлено зв’язок SRA1 rs801460-варіанту з розвитком проліферативного типу доброякісної дисплазії молочної залози з атипією як до, так і після виконання поправки на фактори ризику (вік, індекс маси тіла, вік менархе, прийом оральних контрацептивів та обтяжений анамнез щодо раку молочної залози). Ризик виникнення цього захворювання для rs801460 TT-генотипу є у 2.2 рази вищим (довірчий інтервал 1.010–4.800; p = 0.047), ніж для осіб із CC- and CT-генотипами. У той же час не було виявлено зв’язку між SRA1 rs10463297-варіантом та розвитком проліферативного типу доброякісної дисплазії молочної залози з атипією серед українських жінок. Висновок: У цьому дослідженні випадок-контроль показано, що саме SRA1 rs801460, а не rs10463297, може бути генетичним предиктором розвитку доброякісної дисплазії молочної залози з атипією серед українських жінок.
Ключові слова: доброякісна дисплазія молочної залози, SRA, однонуклеотидний варіант.
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