Relationship between endometriosis and vaspin RS2236242 gene polymorphism

Summary

Objective: The aim of this study is to investigate the relationship between endometriosis and the vaspin RS2236242 gene polymorphism. Materials and methods: This prospective cross-sectional case-control study included patients with grade 4 endometriosis and a healthy control group. Vaspin RS2236242 gene polymorphism was evaluated in these study groups. Results: Thirty eight endometriosis individuals and 17 women from the control group in the study. The group of individuals with endometriosis exhibited similar characteristics to the control group in terms of sex, body mass index (BMI), and age (control mean age: 29.6 ± 4.62 years; BMI: 24.02 kg/m²; endometriosis mean age: 30.4 ± 5.01 years; BMI: 23.63 kg/m²). According to the statistical analysis, there was a significant difference in the genotype distribution of the vaspin RS2236242 polymorphism between people with endometriosis and controls (P = 0.027). Also, the AT genotype was more likely to cause endometriosis than the OR: 2.474 (95% Cl 0.668–9.169) genotypewhen we looked at the genotypes‘ relative risk ratio for endometriosis. Significant differences were observed in total AT and TT genotype frequencies between cases and controls (OR = 2.31; 95% CI 0.86–0.92; P = 0.03). AT and TT genotypes were associated with endometriosis risk. Conclusion: A significant association was observed between vaspin RS2274907 A/T polymorphism and the probability of developing endometriosis.

Keywords:

Endometriosis – SNP – DNA isolation – vaspin RS2274907

Introduction

Endometriosis is a benign gynecological dis ease affecting approximately 6–10% of cases in women of reproductive age [1]. Endometriosis is a complex dis ease resulting from estrogen-dependent, chronic inflammatory conditions. Inflammation caused by the effects of estrogen through the estrogen receptor b is the underlying main process [2]. Although endometriosis affects primarily pelvic organs and ovaries, it is a systemic dis ease. The exact molecular and pathophysiological pathways of dis ease are not clearly determined. It was revealed that heredity has a 50% effect in twin studies, so the etiology of endometriosis may be related to genetic factors [3]. Genome-wide association studies (GWASs) have helped us learn more about the genetic and epigenetic aspects of endometriosis. These studies found that single nucleotide polymorphisms (SNPs) were strongly linked to the risk of get ting endometriosis [4,5].

Vaspin is a recently discovered adipokine that is released from visceral adipose tissue. It consists of 415 amino acids [6,7]. This substance belongs to the group of serine protease inhibitors, and its levels in the blood are linked to inflammation. Scientists have discovered that this adipokine has anti-inflammatory, anti-migratory, and anti-apoptotic properties on vascular endothelial and smooth muscle cells [8,9]. The vaspin gene is situated on chromosome 14q32.13 and is composed of 6 exons and 5 introns [10].

The vaspin gene contains two intronic polymorphisms, specifically intron 2 RS77060950 G/T and intron 4 RS2236242 A/T, which have been linked to various dis eases, including polycystic ovary syndrome (PCOS) [11], type 2 diabetes mellitus metabolic syndrome, obesity [12,13], Coronary Artery Dis ease (CAD), and Nonalcoholic Fatty Liver Dis ease (NAFLD) [14].

Our objective was to examine the distribution of genotypes and high-risk alleles of vaspin in endometriosis. In addition, we also performed the correlation of genotype and stage of endometriosis with various parameters to understand the relationship between them.

Materıals and methods

Working population

The study we conducted was a cross-sectional case-control study, which included 43 individuals with endometriosis and 17 control cases. Since DNA isolation could not be performed in five cases, the study was continued with 17 controls and 38 endometriosis cases. The study was carried out in the gynecology and obstetrics clinic of Selcuk University Medical Faculty between November 20, 2019 and May 20, 2020. In this study, all procedures involving participants complied with the ethical standards of the Selcuk Medical Sciences University ethics committee, the Helsinki Declaration, and the ethical standards of 1964. This research was sup ported by Selcuk University Scientific Research Projects Coordination with the project number 19102061. All individual participants who took part in the study provided informed consent.

The stage of the dis ease in endometriosis cases was assessed using the new American Society for Reproductive Medicine categorization system (ASRM 1996). The study exclusively selected women dia gnosed with stage IV endometrioma.

Blood from patients with endometriosis and control cases was collected in an EDTA anticoagulant tube and stored at –20 °C until genotyping.

Reagents used

DNA isolation

High Pure PCR Template Preparation Kit Cat. No. 11 796 828 001 (ROCHE brand) was used.

Vial/cap label contents/function is described below.

1.
White: Tissue Lysis Buffer is a solution containing 20 mL of the fol lowing components: 4 M urea, 200 mM Tris, 20 mM NaCl, and 200 mM EDTA, with a pH of 7.4 at 25°C.

2.
Green: Binding Buffer • The solution consists of 20 milliliters of a mixture containing 6 molar guanidine-HCl, 10 millimolar urea, 10 millimolar Tris-HCl, and 20% Triton X-100 (volume/volume), with a pH of 4.4 at 25 degrees Celsius.

3.
Pink: Proteinase K. The product is a lyophilized recombinant PCR-grade substance that is used for sample lysis and the inactivation of endogenous DNase.

4a.
Black: Inhibitor Removal Buffer • Combine 33 ml with an additional 20 mL of 100% ethanol. This will result in a final concentration of 5 M guanidine-HCl and 20 mM Tris-HCl at a pH 6.6 (25°C) when the ethanol is added.

4b.
Blue: Wash Buffer • Add 20 ml of 100% ethanol, then 80 ml. • After the addition of ethanol, the final concentrations of NaCl and Tris-HCl will be 20 mM and 2 mM, respectively, with a pH of 7.5 at 25°C.

5.
Colorless Elution Buffer • The solution contains 40 mL of Tris-HCl solution with a concentration of 10 millimolars and a pH of 8.5 at 25 degrees Celsius. Filter tubes with a high level of purity. There are two bags containing 50 polypropylene tubes, each of which has two layers of glass fiber fleece. These tubes may hold a sample volume of up to 700 μL. There are eight bags containing 50 polypropylene tubes, each with a volume of 2 mL.

Before you start the experiment

The dry heat block is brought up to 70 °C.

The oven is brought up to 37 °C.

Proteinase K is put into 4.5 mL distilled water proteinase K bottle (pink cap). This solution is cut into small pieces and divided into Eppendorf tubes and stored at –15/–25 °C. It is dissolved by removing the appropriate amount.

Inhibitor removal buffer is put into a 20 mL pure ethanol inibitor removal buffer (black cap) bottle and mixed gently. Thus, the solution is ready.

Wash buffer is put in an 80 mL pure ethanol wash buffer bottle (blue cap) and mixed gently. Thus, the solution is ready.

Two elution buffers are put into 1.5 mL Eppendorf tubes and placed in a dry heat block at 70 °C for later use.

Necessary materials

1-İsopropanol.

Other materials required

96–100% ethanol (purity suitable for molecular bio logy use):

sterile dH₂O;

heater block or water bath;

vortex device;

microcentrifuge;

microcentrifuge tubes (1.5 mL);

micropipette set and filter micropipette tips.

Procedure

1.
Sample + 200 μL. Binding Buffer (green cap) + 40 μL. Put in proteinase K and set it at 70 °C.

Meanwhile, the number of patients in an Eppendorf tube was put in the X200 μL elution buffer (colorless c).

1.
Sample + 200 μL Binding Buffer (green cap) + 40 μL. Put in proteinase K and set it at 70 °C. Capped tube is heated to 70 °C.

2.
For samples over 100 μL, add isopropanol and mix. Put the sample number on the bottom of the filtered tube; the lid of the filtered tube was closed, and the number was written on it.

3.
Tubes containing this combination were placed in a centrifuge and subjected to a centrifugal force of 8,000 g for 1 min. The lower tube was discarded, and was replaced with a new tube.

4.
Filter tube. The inhibitor removal buf fer (contained in a tube with a black top) was placed upside down and then subjected to centrifugation with a force of 8,000 times the acceleration due to gravity for 1 min. The lower tube was discarded, and was replaced with a new tube.

5.
500 μL was put into the filter tube. Wash buffer (blue-capped tube) was placed and centrifuged at 8,000 g for 1 min by overturning. The bottom tube was discarded, and was replaced with a clean tube.

Repeat steps 6–5, then pour the liquid into the bottom tube, place it under the repeating filter tube, and re-centrifuge at 13,000 Xg at maximum speed.

7.
Normal 1.5 mL solution. The Eppendorf tube was taken, and labeled with a number written on the cap. The filtered tube was placed in this Eppendorf tube. Over 200 μL solution. The heated elution buffer (colorless cap tube) was placed at 70 °C and centrifuged at 8,000 × g for 1 min (this stage was 2 × 200).

Primer and probe design

All amplification primers are standard phosphoramide chemistry (MWG-Biotech) and all VIC/FAM-labeled probes are synthesized by Thermo Fisher and purified by reverse-phase HPLC. One set of primary probes was used for vaspine in amplification. Primary probes marked VIC/FAM were used. Nucleotide A was detected with the VIC probe, and the T nucleotide was detected with the FAM probe.

PCR conditions

Concentration and quantities of materials used for the RS2236242 analysis are given in Tab. 1, while PCR conditions for polymorphism analysis are given in Tab. 2.

Genotype determination (end point analysis)

VIC and FAM hydrolysis probes were used in genotype determination, which was done by using the binding feature of the probes. One probe binds to nucleotide A, and the other binds to T nucleotide. End-point analysis makes use of this difference and distinguishes between natural and mutant types.

Five cases were excluded from the study because DNA isolation could not be performed.

Statistical analyses

Statistical analyses were performed using SPSS 18.0 software.

Hardy-Weinberg analysis was performed to compare the observed and expected genotype frequencies using the c² test. We used odds ratios (ORs) and 95% confidence intervals (CIs) from a logistic regression analysis to figure out the link between vaspin RS2236242 genotypes and the risk of endometriosis (P < 0.05 was considered statistically significant).

Results

Thirty eight endometriosis individuals and 17 control groups participated in the study. The group of individuals with endometriosis exhibited similar characteristics to the control group in terms of sex, body mass index (BMI), and age (control mean age: 29.6 ± 4.62 years; BMI: 24.02 kg/m²; endometriosis mean age: 30.4 ± 5.01 years; BMI: 23.63kg/m²).

DNA isolation of all cases was performed except for 5 cases, and the end point analysis of all cases is given in Fig. 1. According to the statistical analysis, there was a significant difference in the genotype distribution of the vaspin RS2236242 polymorphism between people with endometriosis and controls (P = 0.027). Also, the AT genotype was more likely to cause endometriosis than the OR: 2.474 (95% CI 0.668–9.169) genotype when we looked at the genotypes‘ relative risk ratio for endometriosis (Tab. 2).

Significant differences were observed in total AT and TT genotype frequencies between cases and controls (OR = 2.31; 95% CI 0.86–0.92; P = 0.03). AT and TT genotypes were associated with endometriosis risk.

However, the TT genotype was found to be two times more common in people with endometriosis (N = 20) than in people without endometriosis (N = 11) (52, 63, etc., 64.70% rates within their group). There was also a tendency for a more frequent prevalence of the AT genotype in individuals with endometriosis (N = 18) compared to the control group (N = 4) (47, 36, etc., 23.52% rates within their group). The AA genotype was only found in the control group (N = 2) (Fig. 2).

Discussion

In the present study, the relationship between the vaspin gene RS2236242 polymorphism and endometriosis was investigated. In our study, which is the first research on this subject in the literature, a significant relationship was found between the vaspin gene RS2236242 polymorphism and endometriosis.

Endometriosis is a benign gynecological dis ease affecting approximately 6–10% of cases in women of reproductive age [1]. It is a systemic dis ease that occurs with ectopic endometrial cell growth outside the uterus [15,16]. Endometriosis causes inflammation, resulting in pain, alteration of nearby tissues, fibrosis, the creation of adhesions, and infertility. Although it is believed that the immune system and inflammation are the causes of the dis ease, the precise molecular and pathophysiological mechanisms are still unknown [17,18].

Genome-wide association studies (GWASs) have helped us learn more about the genetic and epigenetic aspects of endometriosis. These studies found that single nucleotide polymorphisms (SNPs) were strongly linked to a person‘s likelihood of getting endometriosis [4,5]. In previous GWAS studies, ten genome-wide SNPs were detected in cases of advanced endometriosis in the Caucasian race [19–25]. Many of these genes were associated with breast and ovarian cancer in addition to endometriosis [26,27].

Adipokines generally function in cases such as inflamation, cell division, cell differentiation, tumor development, and metastasis [28]. Vaspin is an adipokine secreted by visceral adipose tissue, and has 415 amino acids [6,7]. This substance belongs to the group of serine protease inhibitors, and its levels in the blood are linked to inflammation. There isn‘t a lot of information about what vaspin does, but this adipokine has been shown to help vascular endothelial and smooth muscle cells by reducing inflammation, stopping migration, and stopping cell death [8,9]. Studies have shown that in isolated adipocytes, vaspine attenuates the inflammatory cytokine response triggered by IL-1 by inhibiting the NFkB pathway [29].

In our study, we found a significant difference in the frequency of the intronic polymorphism RS2236242 A/T distribution between women with endometriosis and control groups. There were notable disparities in TA genotype frequencies between individuals with the condition and those without it (OR = 0.59; 95% CI 0.37–0.59; P= 0.03). The current study found that having the A allele was linked to a lower risk of getting endometriosis (OR = 0.67; 95% CI 0.46–0.96; P = 0.03), stopping the dis ease from getting worse. The difference between AT + AA and TT genotypes is that individuals with AT + TT genotypes had a greater risk of developing endometriosis (OR = 0.58; 95% CI 0.37–0.92; P = 0.02). SNP changes in the vaspin gene detected up until now may have much more value than is known in the dis ease development [10,30].

Therefore, our study opens up a new area to understand the role of vaspin in endometriosis. This study is the first to establish a connection between the vaspin RS2236242 A/T polymorphism and endometriosis. However, similar to endometriosis, comprehensive research involving a substantial number of patients is necessary to investigate the vaspin gene and other chronic inflammatory conditions.

Conclusion

Our findings suggest a significant correlation between the vaspin RS2274907 A/T polymorphism and the probability of developing endometriosis.