Investigation of VEGF gene polymorphism rs 35569394 in endometriosis

Introduction: Endometriosis is a chronic gynecological condition characterized by the presence of endometrial tissue in locations outside the uterus. Its symptoms include dysmenorrhea, dyspareunia, chronic pelvic pain, infertility, dysuria, and dyschezia. Several studies have related the vascular endothelial growth factor (VEGF) gene polymorphisms as a factor for the development of endometriosis. Objectives: The aim of this study was to compare the frequency of the VEGF gene polymorphism rs35569394 in women with endometriosis and a control group, as well as to investigate the association of this polymorphism with the risk of endometriosis. Methods: This case-control study included 45 endometriosis patients (cases) and 78 controls. The molecular analysis was performed by polymerase chain reaction (PCR). The chi-square test was used to compare the genotype and allele frequencies, and the Hardy-Weinberg equilibrium was also tested using the chi-square test, considering the significance of p < 0.05. Results: The frequency of the II, ID and DD genotype was 24.4% versus 26.9%; 35.6% versus 37.2%; and 40% versus 35.9%, in patients and the control group, respectively. The allele frequencies were I: 0.42 (cases) and 0.45 (control); D: 0.58 (cases) and 0.55 (control). The allele and genotype frequency of VEGF -2549I/D polymorphism was not different between women with endometriosis and the control. Conclusion: In the present study, the frequency of the VEGF gene polymorphism rs35569394 is similar between cases and control. This genetic variant is not associated with endometriosis in the investigated population.


introDuCtion
Endometriosis is a chronic gynecological condition characterized by the presence of endometrial tissue in ectopic locations.Its symptoms include dysmenorrhea, dyspareunia, chronic pelvic pain, infertility, dysuria and dyschezia (1) .Due to the symptomatology, patients usually exhibit high levels of depression, anxiety (2) and sexual dysfunction (3) .
Endometriosis presents characteristics similar to neoplasia, since neovascularization is needed for the implantation and development of endometrial tissue (4) and therefore, angiogenesis regulating genes, such as vascular endothelial growth factor (VEGF), may be related to the development of the disease.A recent review (5) presents an overview of the basic mechanisms of vascularization in endometriosis, with particular emphasis on its future clinical implications in the diagnosis and therapy of the disease.The authors conclude that the establishment of approaches based on vascularization in the management of endometriosis is still a great challenge.For diagnostic purposes, reliable angiogenic and vasculogenic biomarkers with high sensitivity and specificity must be identified (5) .
The VEGF gene is highly polymorphic, and several studies have related the polymorphisms in this gene as a factor for the development of endometriosis (4) .An insertion/deletion (I/D) polymorphism was identified at position -2549 of the promoter region of the VEGF gene (rs35569394) and is characterized by an 18 base pairs (bp) deletion at that region (D allele).This genetic variant has been studied and related to several conditions such as Behçet's disease (6,7) , renal disease (8) , breast cancer (9) , diabetic retinopathy (10) , diabetic nephropathy (11) , recurrent spontaneous abortion (12) , preeclampsia (13) and uterine fibroids (leiomyomas) (14) .However, this polymorphism was not associated with hepatocellular carcinoma (15) , recurrent miscarriage (16) , essential hypertension (17) and systemic lupus erythematosus (18) .However, we are not aware that studies on the relationship of this polymorphism with endometriosis have been carried out.
It is worth mentioning that a recent review has shown that there is a growing and significant scientific production on the role of genetic polymorphisms or single nucleotide polymorphisms (SNPs) in the etiology of endometriosis, stating their participation in the development of this condition (19) .Considering the assumption and the numerous scientific evidence of the contribution of the VEGF gene to the pathogenesis of endometriosis, the present research is justified.

oBjECtivES
The aim of this study was to compare the frequency of the VEGF gene polymorphism rs35569394 in women with and without endometriosis, as well as to verify its association with this condition.

MEthoDS
This project was approved by the Research Ethics Committee [Comitê de Ética em Pesquisa (CEP)] of the Universidade Federal do Triângulo Mineiro (UFTM) -protocol no.1628.Forty-five samples of women with endometriosis (age ranged from 24 to 58 years) and 78 samples of women without endometriosis (age ranged from 22 to 70 years) from the Gynecology and Obstetrics Outpatient Clinic of the UFTM were evaluated.The inclusion criteria were that the woman must has performed laparoscopy or laparotomy that allowed the confirmation of the presence of endometriosis (patients) or their absence (control), and to voluntarily participate in the study by signing the informed consent form.Women who did not meet the criteria previously described were excluded.The period for data collection was 18 months.
The main surgical indications were chronic pelvic pain, dysmenorrhea and dyspareunia.According to the criteria established by the American Society of Reproductive Medicine (20) , 47% of women exhibited stages III-IV (moderate-severe endometriosis) and for 38% of patients, this information was not available in the medical record.Only 15% of the patients exhibited minimal-to-mild endometriosis (stages I-II).In the present study, laparoscopy combined with histological confirmation was the standard method for diagnosis of endometriosis.
Participants' deoxyribonucleic acid (DNA) was extracted from peripheral blood according to the technique described by Miller et al. (1988) (21) , quantified in spectrophotometer and amplified by polymerase chain reaction (PCR).
The polymorphism rs35569394 in the promoter region of the VEGF gene was amplified using the following primer sequence: sense primer: 5' GCT GAG AGT GGG GCT GAC TAG GTA 3' and antisense primer: 5' GTT TCT GAC CTG GCT ATT TCC AGG 3'.PCR conditions were 1× buffer, 2 mM MgCl2, 0.2 uM of each deoxyribonucleotide triphosphate (dNTP), 20 pmols of each primer, 1 U of Taq DNA polymerase and approximately 100 ng of DNA.Amplification occurred with initial denaturation at 95°C for 10 minutes, followed by 35 cycles with denaturation at 95°C for 45 seconds, annealing at 60°C for 45 seconds and extension at 72°C for 45 seconds, and finally extension at 72°C for 10 minutes.The deletion of 18 bp allele produces a 211 bp fragment, and the insertion, a 229 bp band, that is, three genotypes will be possible: DD (211 bp band), II (229 bp band) and ID (two bands, one of 211 bp and one of 229 bp).
For the determination of the genotypes, electrophoresis was performed on 5% polyacrylamide gel stained with silver nitrate.The staining steps were: 1. fixation solution (83.3 ml of ethanol; 3.33 ml of acetic acid, distilled water sufficient for 500 ml qsp for 10 minutes; 2. silver solution (1 g silver nitrate; distilled water 500 ml qsp) for 10 minutes; 3. two quick washes with distilled water; 4. developer solution (15 g NaOH; 3.33 ml formaldehyde; distilled water 500 ml qsp) for 5 minutes; and 5. storage in fixation solution.
Statistical analysis used the chi-square test for comparison of genotype and allele frequencies; the Hardy-Weinberg equilibrium (HWE) was also tested using the chi-square test, considering significance of p < 0.05.The statistical power obtained was 85%, using the GPower program.

rESuLtS
The distribution of genotypes and allele frequency in patients and controls is shown in the Table.
Statistically, there was no significant difference between the two groups regarding the distribution of genotype (c 2 = 0.216; p = 0.90) and allele (c 2 = 0.325; p = 0.57) frequencies of the VEGF gene polymorphism -2549 I/D in patients and control, respectively.The distribution of the genotype was in the Hardy-Weinberg equilibrium in patients (c 2 = 3.31; p = 0.07), but not in the control group (c 2 = 4.89; p = 0.03).
The Board compares the genotype and allele frequencies of our study with those published in the literature that did not associate the polymorphism with the conditions investigated.
According to the literature, there were no deviations of the HWE among the studied groups (9,10,12,13,15) , disagreeing with the present study, which showed deviations in the control group.HWE is used to check if frequencies remain unchanged over generations.HWE deviations suggest that there is, for example, some sort of selection acting in the population, or that there is migration, or inbreeding, or these phenomena associated (25) .
The present study was presented as a limitation to the casuistry.It is worth noting that the gold standard for the diagnosis of endometriosis is laparoscopy, a surgical procedure performed in view of strong clinical suspicion.This factor interferes significantly in obtaining a larger sample size.However, there are recent studies published in the literature on genetic polymorphisms in endometriosis that investigated 52 (26,27) and 54 (28) patients versus 42 (26) , 46 (28) and 63 (27) control.In our study, 123 women (45 patients and 78 control) were investigated, and the statistical power obtained guarantees the reliability/robustness of the results found.
Further studies of this SNP in other populations are suggested to clarify the real contribution of the VEGF gene polymorphism rs35569394 to the etiology of endometriosis.However, it is noteworthy that this scientific study is pioneering on this polymorphism in endometriosis.

ConCLuSion
In the present study, the frequency of the VEGF gene polymorphism rs35569394 is similar between cases and control.This genetic variant is not associated with endometriosis in the investigated population.

taBLE −
Genotype and allele frequency of study and control groups VEGF: vascular endothelial growth factor.