Polymorphisms in the 3′UTR Region of ESR2 and CYP19A1 Genes and Its Influence on Allele-Specific Gene Expression in Endometriosis

Objectives. Endometriosis is supported by hormonal, immunological, and environmental factors. No specific marker for endometriosis has yet been identified. ESR2 and CYP19A1 genes play a major role in the hormonal control of endometriosis women, the development of which largely depends on steroid hormones. Aim. An analysis of ESR2 and CYP19A1 allele-specific gene expressions in the context of the risk for endometriosis occurrence. Methods. The study material included paraffinembedded tissue specimens, collected from patients (n = 100) with endometriosis. Blood samples from age-matched, endometriosis-free women (n = 100) served as a control. the RT-PCR technique was performed to observe the expression of ESR2 and CYP19A1 genes. Moreover, Sanger’s sequencing method was applied for polymorphism analysis. Results. A set of 4 single-nucleotide polymorphisms (SNPs) was determined; all of them most significantly associated with endometriosis: rs4986938 (G>A)(chromosome 14), rs928554 (A>G) (chromosome 14), rs10046 (C>T) (chromosome 15), and rs4646 (C>A) (chromosome 15). There were no differences in the distribution of genotypes and alleles in the studied groups, taking into account ESR2 and CYP19A1 gene expressions. Conclusion. The ESR2 and CYP19A1 polymorphisms may not be correlated with endometriosis susceptibility. Further analysis is needed to specify the role of these polymorphisms in the pathogenesis of endometriosis.


Introduction
Endometriosis is an oestrogen-dependent disease affects 10-15% of women in the reproductive age and 35-50% of women with pains in the pelvis minor and/or with infertility [1]. Aetiology of the disease is complex. Hormonal, immunological, and environmental factors are responsible for its formation [2][3][4]. In recent years, special attention has been paid to the genetic mechanisms that might have a significant impact on the increased incidence of endometriosis. CYP19A1, aromatase encoding gene, is an enzyme involved in biosynthesis of oestrogens. Different expression levels of aromatase were determined in the endometriosis foci in comparison to the eutopic endometrium. The exact basis of observed changes remains unknown [5].
Endometriosis lesions, unlike the eutopic endometrium, demonstrate some activity of the aromatase [6].
The activity of oestrogens on target cells is possible via oestrogen receptors. Oestrogen receptors (ERs) acting as transcription factors play a significant role in the growth and differentiation processes of endometrial cells, as well as in numerous biological functions in the eutopic endometrium and endometriosis. The two following types of oestrogen receptors have thus far been identified: ERα and ERβ, encoded by two different genes (ESR1 and ESR2, respectively). ERβ is the predominant isoform in patients with endometriosis [7,8].
Different expression levels of aromatase were determined in the endometriosis foci in comparison to the eutopic endometrium. [9].
Previous studies have been demonstrated that both ERs are expressed in human endometriotic tissues but it has been shown that there are significantly increased levels of oestrogen receptor ERβ and decreased levels of ERα in the endometriotic lesions in comparison with the eutopic endometrium. [10][11][12].
The literature data showed that a number of specific SNPs in the 3 ′ UTR regions have structural consequences that may result in the emergence of phenotypic manifestations in the form of the disease [13]. 3 ′ UTR fragments are within the regulatory elements of genes and play an important role in the translation and distribution of RNA.
The reported study was aimed at finding out whether there were any correlations between the allele-specific gene expression of the ESR2 and CYP19A1 genes and the incidence of endometriosis.
The goals of the study included (a) an analysis of the ESR2 and CYP19A1 gene expression levels in patients with endometriosis and in a control group, (b) an analysis of the ESR2 and CYP19A1 gene polymorphisms in the 3′ UTR region in patients with endometriosis and in a control group, (c) a correlation of the assayed levels of the ESR2 and CYP19A1 gene expressions with polymorphic variants, and (d) the assessment of the significance of obtained results in the context of the risk for endometriosis.

DNA Isolation.
Genomic DNA was prepared, using a QIAamp DNA FFPE Tissue Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instruction. DNA was extracted from the blood, using a commercially available QIAmp DNA purification kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instruction.

Detection of Polymorphisms in the ESR2 and CYP19A1
Genes. A reaction mixture of the following composition was used for PCR: 2.5 μl of 10x concentrated PCR buffer, 1 μl of each primer, 0.2 μl of Taq polymerase with 5 U/μl activity, 1 μl of a 200 μM mixture of nucleotides (dNTPs), and 100 ng matrix. The reaction mixture was made up to a final volume of 25 μl. The primers were designed using the Primer3 program (Table 2). For all primer pairs, the amplification reactions were carried out using the following thermal  Real-time PCR was carried out in a Mastercycler® ep realplex device (Eppendorf, Germany). The thermal profile of the reaction included a preliminary denaturation in the temperature of 95°C for 10 minutes, followed by 50 cycles of 15second incubation in 95°C, combined with 1 minute in 60°C. The following, commercially available kits of probes and starters were applied in the real-time PCR: Hs01100353_m1 for ESR2 gene, Hs00903411_m1 for CYP19A1 gene, and Hs02800695_m1 for HPRT1 (hypoxanthine phosphoribosyltransferase 1) gene, being a reference gene. The yield and quality (260/280 optical density ratios) of the RNA products were determined using PicoDrop spectrophotometer (Picodrop Limited, Hinxton, UK) ( Figure 1). The purified total RNA was immediately used for cDNA synthesis or stored at -80°C until use.

Statistical
Analysis. The obtained results were statistically processed by means of STATISTICA 11 software (StatSoft, Poland). The significance of differences was analysed at the level of gene expression and mRNA, using nonparametric tests (the Mann-Whitney U test and the Kruskal-Wallis test) for a lack of distribution normality of the obtained results, as confirmed by the Shapiro-Wilk test. The R Spearman test was used to assess correlations between variables. Statistical analysis of the distribution of genotypes and alleles in the test and control group was carried out after prior confirmation that the obtained systems remain in a state of equilibrium according to the rules of Hardy and Weinberg. Wild type of the genotype and allele was the reference group. The result was
However, the current study failed to show any correlation of analysed SNPs with the expression of the ESR2 and CYP19A1 genes.
The genotype frequency of the ESR2 rs4986938 polymorphism in relation to its expression is summarized in Table 2. There were no statistically significant differences in the ESR2 gene expression in relation to the occurrence of rs4986938 polymorphism (Kruskal-Wallis test p = 0:46).

Disease Markers
There were no statistically significant differences in the CYP19A1 gene expression in relation to the occurrence of rs10046 polymorphism genotypes (Mann-Whitney test p = 0:23) (Table 4).
No relationship was revealed between ESR2 and CYP19A1 polymorphisms and rASRM (revised American Society for Reproductive Medicine) classification scores in the study group. The SNPs in the study group did not correlate with either the age of the patients or with their BMI, menopausal status, or with the number of pregnancies in history.

Discussion
Our research addressed the role of allele-specific gene expression of ESR2 and CYP19A1 genes as a risk factor for endometriosis. Performed genetic analyses were based on the rs4986938, rs928554, rs10046, and rs4646 polymorphisms of the ESR2 and CYP19A1 genes.
The allele-specific gene expression was assayed in patients with endometriosis vs. a healthy control group, while also studying their effects on the increased prevalence of endometriosis among Polish women.
Regarding studies of CYP17 and ESR2 gene polymorphisms, there are few reports concerning the 3 ′ UTR region, which is important, because it attaches to the miRNA, which regulates gene expression. This region is therefore important for phenotypic changes that may trigger development of endometriosis While SNPs in CYP19A1 have been associated with sexsteroid hormone levels and other oestrogen-dependent diseases including endometriosis, the results of previous studies of the associations between various CYP19A1 polymorphisms and endometriosis have been inconsistent up to a certain degree. Many studies of CYP19A1 polymorphisms in relation to endometriosis risk have been published; however, their findings were not confirmed by any additional researches on affected populations [27,28].     Tempfer et al. reported that the ERβ gene is associated with increased risk of stage IV endometriosis in Japanese women, while we found polymorphism in all stages of classification of endometriosis of the patients (classification of endometriosis according to the American Society for Reproductive Medicine) [29].
In the other studies, it was found that the allelic frequency of RsaI polymorphism of the ERβ gene in AG is about nine times higher in patients with endometriosis in comparison to control groups [30].
However, when comparing fertile and infertile subgroups, no significant differences were stated. The study of ERβ and its correlation with the risk of endometriosis could help in the explanation of disease's genetic aetiology.
Szaflik et al. identified statistically significant correlations between new SNP, rs4986938 and rs928554, not described earlier, and endometriosis. In the case of rs4986938, the AA genotype decreased the risk of endometriosis. A similar effect was demonstrated in the case of rs928554 polymorphism of the AG genotype. The results, obtained during that analysis, demonstrated that rs4986938 and rs928554 polymorphisms of the ESR2 gene are associated with the occurrence of endometriosis [31].
We did not find a relationship between allele-specific genes expression and risk of endometriosis. This study is limited by the sample sizes, and it is possible statistical power may be not enough to detect smaller expression differences among the comparison groups. The study results will be useful for further study design with larger samples sizes.
Therefore, further studies would be justified and needed to reveal the common involvement mechanisms of ESR2 and CYP19A1 SNPs in endometriosis formation.
However the results, obtained in the study, contribute to better knowledge of and information on the molecular mechanisms which support the development of endometriosis.

Conclusions
There are no statistically significant differences in the ESR2 and CYP19A1 allele-specific gene expressions based on the comparisons of the 100 cases vs. the 100 controls, which lead us to conclude that the two genes do not play significant roles in the pathogenesis of endometriosis.

Data Availability
The datasets supporting the conclusions of this article are included within the article.

Ethical Approval
This work was supported by the Institute of Polish Mother's Memorial Hospital, Lodz, Poland, from the Statutory Development Fund. All procedures performed in studies involving human participants are in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Consent
All the study participants gave a written informed consent. A formal consent was also issued by the Bioethical Committee of the Institute of the Polish Mother's Memorial Hospital in Lodz (Approval number, 8/2016).

Conflicts of Interest
The authors declare no conflict of interest with regards to the reported study.