In this study we aimed to investigate the significance of SNPs of the VEGF gene (rs699947 (-2578C>A), rs1570360 (-1154G>A) rs2010963 (+405G>C), rs3025039 (+936C>T), rs25648 (c534C>T) in HMB. We have found increased risk for HMB in patients with CC, AC+CC genotype, and C allele of the -2578C>A polymorphic region and AA genotype and A allele of the -1154G>A polymorphic region (p < 0.05). However, we have not observed any associations between HMB and other three candidate polymorphisms of +405G>C, +936C>T, and c534C>T (p > 0.05).
In the literature, we have not encountered any studies emphasising on the relationship between SNPs and HMB. Nevertheless, there are several studies investigating the effect of VEGF gene polymorphisms in cardiovascular diseases, essential hypertension, certain cancer types, psoriasis, and endometriosis. Although being a unique study represents strength of our work, it also enabled us to discuss or compare our results with similar papers. Therefore, we compared our findings with articles about endometriosis, which defines a gynecologic disease that endometrial tissue is present outside the uterus.
Perini et al. [13] evaluated -2578C>A, −460T>C, −1154G>A, +405G>C, and +936C>T polymorphisms in 182 Brazilian women with endometriosis and reported that variant allele -1154A was had significant relationship with endometriosis. They found AA genotype in 9.3% of patients and 1.9% of controls while the frequency of GA+AA genotype was 44.1% in patients and 30.2% in controls, and frequency of A allele was 26.7% in patient group and 16.0% in controls (p < 0.05). In our study, the AA genotype was found in 56.9% of patients and 38.5% of controls while the frequency of GA+AA genotype was 95.4% for patients and 87.7% for controls and the frequency of A allele was 76.1% in HMB patients and 61.5% in controls (p < 0.05). Perini et al. [13] reported an approximate two-fold increased risk for endometriosis in individuals with GA+AA genotype of the -1154G>A polymorphic region and an approximate 6-fold increased risk for AA genotype. Similarly, we have observed an approximate two-fold increased risk for HMB in individuals with GA+AA genotype (p > 0.05), an approximate 3.5 fold increase in AA genotype (p = 0.06), and an approximate two fold increase in case of harbouring A allele (p = 0.04). Although these two studies presents distinct genotype and allele frequencies, individuals having A allele in 1154G>A polymorphic region have been found to have significantly increased risk for the defined diseases.
On the other hand, in another study from Northern China on 344 women with endometriosis and 360 healthy controls by Liu et al. [14], AA genotype of the -1154G>A polymorphic region was alleged to decrease the risk of endometriosis. The frequency of AA genotype of the -1154G>A polymorphic region was 1.7% in patients and 5.8% in controls while the frequency of harboring A allele was 16.1% in patients and 22.2% in controls concluding a statistically significant reduction of endometriosis risk in case of A allele of -1154G>A polymorphism.
Perini at al. [13] did not report an association between endometriosis and VEGF gene -2578C>A polymorphisms. Similarly, in our study the frequency of CC genotype of -2578C>A polymorphism was 38.5% in patients and 24.6% in controls, and the frequency of AC + CC genotype was 86.2% in patients and 72.3% in controls. Despite being more frequent in patients, the difference was not statistically significant (p = 0.083). As for the C allele, the frequency was 62.3% in patients and 48.5% in controls which established a statistically significant difference (p < 0.05). Therefore, we have found an approximate 3 fold increased risk for HMB in individuals with CC genotype of the -2578C>A polymorphic region and an approximate 2 fold increased risk in AC+CC genotype and C allele. Also, Liu et al. [14] reported frequencies of AA, CA, and CC genotypes and A allele frequency of -2578C>A polymorphic region in patient and control groups respectively; 3.2% - 8.1%; 32.0% - 36.4%, and 64.8% - 55.6%, 19.2% - 26.3% concluding a statistically significant difference in terms of genotype and allele harbouring (p <0.004, p = 0.002 respectively). Similarly, having C allele has been associated with HMB in our study as in case with endometriosis.
With regards to the +405G>C, +936C>T polymorphic region our results suggest no significant effect on the susceptibility to HMB. It is noteworthy that our results are consistent with Perini’s study [13] and Zhao and colleagues [15] which consist a larger number (958 cases and 959 controls) of Australian women. Similarly Liu et al. [14] found that the + 936C>T polymorphic region does not have a significant relationship with the risk of developing endometriosis.
The main reason for these inconsistent results may be the differences between ethnic groups. For example, the Ensembl database shows that the frequency of the VEGF -2578 A allele was 27.2% with the CHB (Han Chinese in Beijing), 34.1% with AMR (American), and 45.5% with CEU (Utah Residents with Northern and Western European Ancestry).
In the literature, we found two studies from our country evaluating the effect of VEGF gene +405G>C polymorphism on endometriosis risk with controversial results. Altınkaya et al. [16] evaluated the genotype frequencies of VEGF +405G>C polymorphic regions on 98 endometriosis patients and 94 controls. They detected GC genotype in 58.2% of patients and 10.6% of controls and G allele in 45.4% of patients and 5.3% of controls. They suggested that GC genotype and G allele may be associated with the advanced grade endometriosis risk in the Turkish population. In another study from Turkey, Attar et al. [17.] also proposed that CC genotype of +405G>C polymorphisms may be related to endometriosis whereas G allele has a protective role against endometriosis. The small sample sizes impede with reflecting data of the whole Turkish population and may partially explain the conflicting results.
The meta-analyses also yielded conflicting results. In a meta-analysis evaluating 14 eligible studies on a total of 3313 endometriosis patients and 3393 controls, VEGF gene -1154G>A polymorphic region GA genotype and A allele and -2578C>A polymorphic region CC genotype and C allele were found to reduce endometriosis risk whereas +936C>T poymorphic region CT genotype and T allele increased the risk similar to our results on HMB [18]. No effect of the +405G>C polymorphism was reported in line with our findings. On the contrary, Jiang et al. [19] stated that VEGF + 405G>C and + 936C>T SNPs may be associated with the risk of endometriosis in their meta-analysis, in which they investigated 9 studies including 1610 endometriosis patients and 1643 control cases.
The most important limitation of the current study is the small sample size which can also be held responsible for the controversial results in other studies in the literature. Besides, the facts that these studies have been performed on distinct ethnic groups and different diseases and having identified various haplotypes may have led to the conflicting results. These diverse results suggest that disease tendency may stem from not solely a single polymorphism but a combined effect of polymorphisms and that haplotype effect may play a more significant role in disease tendency than single polymorphisms.
In our study in the examination of the -2578C>A, -1154G>A,+405G>C and 534C> T polymorphic regions of the VEGF gene we have identified 4 haplotypes: CACC (38.1%), AGGC (31.2%), CAGC (17.3%), AAGT (12.3%). AGGC haplotype was less prevalent in HMB patients (23.8% vs 38.5%) while CAGC haplotype was more frequent in the HMB group (25.4% vs 9.2%). Perini et al. [13] emphasized that CCGG haplotype derived from four polymorphisms (-2578C>A / -460T>C / -1154G>A / + 405G>C) has a protective effect against the development of endometriosis (3% in cases, 6.7% in the control group). Liu et al. [14] stated that the VEGF gene -460C>T -1154G>A, -2578C>A regions included CAA, TAA and TAC haplotypes can significantly reduce the risk of developing endometriosis, and the CAC haplotype can significantly increase the risk of developing endometriosis. The polymorphic regions included in the studies produced different linkage disequilibrium results and different haplotypes were obtained in each study making it difficult to compare study results.
Our study on VEGF gene polymorphisms in Turkish women with HMB suggests increased risk for C allele, CC, and AC+CC genotypes of −2578A>C polymorphic region, A allele, AA genotype, and CAGC haplotype of the −1154G>A polymorphic region whereas protective role of the AGGC haplotype. However, further studies with a larger sample size are required to figure out a more certain association.