Human Reproduction Vol.20, No.7 pp. 1844–1849, 2005
doi:10.1093/humrep/deh852
Advance Access publication March 3, 2005
The vascular endothelial growth factor (VEGF) 1 405G>C 50-untranslated region polymorphism and increased risk of endometriosis in South Indian women: a case control study Manjula Bhanoori1, K.Arvind Babu1, N.G.Pavankumar Reddy1, K.Lakshmi Rao1, Krina Zondervan2, Mamata Deenadayal3, Stephen Kennedy4 and S.Shivaji1,4,5 1
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India, 2Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK, 3Infertility Institute and Research Centre, Hyderabad, India and 4Nuffield Department of Obstetrics & Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford, UK 5
To whom correspondence should be addressed at: Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India. E-mail:
[email protected]
BACKGROUND: Vascular endothelial growth factor (VEGF), a major mediator of angiogenesis and vascular permeability, is known to play a key role in the pathophysiology of endometriosis. METHODS AND RESULTS: The single nucleotide polymorphisms, 2460C> T and 1405G> C, in the 50 -untranslated region of the VEGF gene were tested for association in a case – control study of 215 affected women and 210 women with no evidence of disease. All the women were of South Indian origin and ascertained from the same infertility clinic. The genotype and allele frequencies of the 2460C> T polymorphism did not differ significantly between cases and controls. In contrast, the genotype (P 5 0.002) and allele (P 5 0.001) frequencies of the 1405G> C polymorphism showed a significant difference between cases and controls. The 1405 GG genotype was found more often in patients with an endometrioma > 3 cm compared to controls. The frequency of the 2460T/1405C haplotype (P 5 0.016) was significantly lower in affected women compared to controls. CONCLUSIONS: The 2460T/1405C haplotype in the VEGF gene, which is associated with lower promoter activity, was significantly less common in women with endometriosis than in controls. These data suggest that the 1405G allele may influence the likelihood of a woman developing the disease. Key words: endometriosis/polymorphism/VEGF
Introduction Endometriosis is a chronic gynaecological disease characterized by the growth of hormonally responsive, endometriallike tissue outside the uterine cavity. Up to 10% of women of reproductive age may be affected (Eskenazi and Warner, 1997). The clinical presentation varies, although affected women usually have one or more pain symptoms and/or difficulty conceiving. Although Sampson’s theory of the transplantation of endometrial tissue onto the pelvic peritoneum via retrograde menstruation is one of the most widely accepted explanations for the development of the disease (Sampson, 1927), the actual cellular and molecular mechanisms responsible are unclear. However, the cause is almost certainly multifactorial involving environmental, immunological, endocrine and genetic processes (Olive and Schwartz, 1993). Association studies have identified mutations and single nucleotide polymorphisms (SNPs) in a number of genes that might confer susceptibility to endometriosis, but their precise role remains to be determined. Histologically, endometriosis is a benign disease but it can behave like a malignancy in terms of growing, infiltrating
and adhering to the surrounding tissues (Varma et al., 2004). To survive and develop into an endometriotic lesion, the ectopic tissue must induce new vessel formation to connect to the vascular system (Nisolle and Donnez, 1997; Taylor et al., 2002). A key mediator in neoangiogenesis is vascular endothelial growth factor (VEGF) as it stimulates endothelial cell proliferation and migration, and increases vascular permeability (Risau, 1997; Ferrara, 1999); it is therefore regarded as a marker of tumour invasion and metastasis (Carmeliet and Jain, 2000). SNPs within the VEGF gene have been identified, some of which have functional significance. For example, the þ 405G. C polymorphism in the 50 untranslated region has a significant effect on VEGF protein production (Brogan et al., 1999; Watson et al., 2000). Association has been reported in case – control studies between VEGF polymorphisms and diseases such as diabetic retinopathy (Ray et al., 2004), prostate cancer (Lin et al., 2003) and breast cancer (Krippl et al., 2003). VEGF may have a pivotal role in the development and progression of endometriosis because: (a) it is expressed in human uterine epithelial and stromal cells and regulated by
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VEGF polymorphism and endometriosis
estrogen (Hyder et al., 1996; Mueller et al., 2000); (b) it is localized in the epithelium of endometriotic implants, particularly in red lesions (Donnez et al., 1998; Tan et al., 2002); and (c) VEGF levels are elevated in the peritoneal fluid and serum of endometriosis patients (McLaren et al., 1996; Taylor et al., 1997; Matalliotakis et al., 2003). VEGF has even been proposed as a diagnostic and therapeutic target in endometriosis (Hull et al., 2003; Bedaiwy and Falcone, 2004; Nap et al., 2004). Recently, the 2 460C. T polymorphism in the 50 -untranslated region of the VEGF gene was shown to be associated with endometriosis (Hsieh et al., 2004). In the present study, we compared the frequencies of the 2 460C. T and þ 405G. C SNPs in the 50 -untranslated region of the VEGF gene in women with and without endometriosis from South India.
taining 50 ng genomic DNA, 2 – 6 pmole of each primer, IX Taq polymerase buffer (1.5 mM MgCl2) and 0.25 U of Amplitaq DNA polymerase (Perkin Elmer, Foster City, USA). The primers for 2460C. T were 50 -TGTGCGTGTGGGGTTGAGCG-30 (forward), and 50 -TACGTGCGGACAGGGCCTGA-30 (reverse), and 50 -ATTTATTTTTGCTTGCCATT-30 (forward) and 50 -GTCTGTCTGTCTGTCCGTCA-30 (reverse) for þ405G. C, using the method described by Watson et al. (2000). PCR amplification was performed in a programmable thermal cycler gradient PCR system (Eppendorf AG, Hamburg, Germany). The PCR amplification was carried out for 30 cycles (denaturation at 948C for 1 min, annealing for 1 min at 608C for 2 460 and 558C for þ405, extension at 728C for 1 min and final extension for 10 min at 728C). PCR products of 175 bp (2 460) and 304 bp (þ405) were analysed by 1.5% agarose gel electrophoresis followed by ethidium bromide staining and ultraviolet visualization and then sequenced with a Taq-Dye deoxy-terminator cycle sequencing kit (Applied BioSystems, Foster City, USA) using an automated ABI 3770 sequencer (Applied BioSystems, Foster City, USA)
Materials and methods
Statistical analysis Statistical analysis was performed using SPSS statistical package (V 11.0). The genotypic distribution amongst subjects was tested for Hardy – Weinberg equilibrium using Fisher’s exact test. The allele ratios and genotype distributions in the cases and controls were analysed with Pearson x 2 or Fisher’s exact test. Odds ratios were calculated together with their 95% confidence intervals (CI). Haplotype frequencies for multiple loci and the standardized disequilibirum coefficient (D0 ) for pair-wise linkage disequilibrium testing were calculated using the Arlequin program (Version 2.000, Genetics and Biometry Laboratory, University of Geneva, Geneva, Switzerland) (Schneider et al., 2000) using the ‘Expectation – Maximization algorithm’ (Excoffier and Slatkin, 1995) and univariate logistic regression was used for haplotype association analysis. All P-values were twotailed and 95% confidence intervals (CI) were given.
Subjects All the subjects were non-smokers and of South Indian origin. Two hundred and fifteen unrelated women with moderate – severe (III – IV) endometriosis staged using the revised American Fertility Society (rAFS) classification system (1997) were recruited at the Infertility Institute and Research Centre (IIRC), Hyderabad, India. All women had a trans-vaginal ultrasound scan (TVS) at screening followed by laparoscopy to confirm the diagnosis (rAFS III ¼ 80; IV ¼ 135). All the patients had different forms of endometriosis such as peritoneal lesions, adhesions and endometrioma. Women with other ovarian cysts, adenomyosis, ovarian cancer, fibroids, and stage I and II endometriosis were excluded from the study. Their mean age ^ SD was 27.5 ^ 4.4 (range ¼ 20 – 40) years. All the patients complained of dysmenorrhoea (mild ¼ 47%; moderate ¼ 30%; severe ¼ 23%) and 75% had dyspareunia. Most women (97.7%) were infertile (primary ¼ 78%; secondary ¼ 22%). Two hundred and ten women were recruited from the same clinic population and had an equal opportunity to be identified as cases, thereby meeting the criteria for appropriate controls set by Zondervan et al. (2002a). The controls consisted of 141 (67%) women with no evidence of endometriosis on TVS and laparoscopy and 69 (33%) women with no evidence of an ovarian endometrioma on TVS (and no clinical symptoms of endometriosis) who therefore did not subsequently have a laparoscopy. Their mean age ^ SD was 26.7 ^ 3.9 (range 22 – 39) years. Seventy-four percent had primary and 26% had secondary infertility. Among the symptomatic controls, 29% complained of mild dysmenorrhoea and 20% complained of dyspareunia. Written, informed consent was obtained from all participants. The Institutional Review Board of the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, approved the study. Determination of the VEGF genotype DNA extraction Genomic DNA was extracted from 1 ml of EDTA anti-coagulated whole blood by the salting out method (Miller et al., 1988). Primers and polymerase chain reaction Genotyping of the 2460C. T and þ405G.C polymorphisms in the 50 UTR of the VEGF gene was determined by PCR and sequencing analysis. PCRs were carried out in a total volume of 25 ml, con-
Results Amongst both cases and controls, the genotypic distributions of the individual SNPs as well as the VEGF allele system were all in Hardy – Weinberg equilibrium (P. 0.05). Sequence analysis of 175 bp (2 460) and 304 bp (þ 405) products of the VEGF 50 -untranslated region SNPs are shown in Figure 1. CC, TT (460) and GG, CC (þ 405) homozygotes manifested as a single peak, whereas heterozygotes TC (2 460) and GC (þ 405) as double peaks (Figure 1A and B). Genotype and allele frequencies for the VEGF 2 460 and þ 405 SNPs are summarized in Table I. The 2 460 genotype frequencies amongst the 215 cases were CC ¼ 21.8%, TC ¼ 52.1% and TT ¼ 26.1%; the C and T allele frequencies were 48% and 52%, respectively. The 2 460 genotype frequencies amongst the 210 controls were CC ¼ 20.0%, TC ¼ 53.3% and TT ¼ 26.7%; the C and T allele frequencies were 46.6% and 53.4%, respectively. There was no statistically significant difference in the genotype distributions or allele frequencies of VEGF 2 460 between the cases and controls. The þ 405 genotype frequencies amongst the 215 cases and 210 controls were CC ¼ 1.9% and 8.6%; GC ¼ 33.0% and 37.6%; GG ¼ 65.1% and 53.8%, respectively. The allele frequencies were C ¼ 18.3% (cases) and 27.3% (controls) 1845
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Figure 1. VEGF 2 460C. T (A) and þ 405G. C (B) polymorphism by sequence analysis of the PCR-amplified product using a forward primer.
and G ¼ 81.7% (cases) and 72.7% (controls). Allele frequencies were significantly different between cases and controls (P ¼ 0.001), which resulted from an increased proportion of homozygote GG genotype carriers (but not heterozygote GC carriers) among endometriosis patients as compared to controls (P ¼ 0.002). We have also analysed the data considering only 141 laparoscopically tested controls and we did not find any variation in the association status with both the SNPs (data not shown). Genotype frequencies for the VEGF 2 460 and þ 405 SNPs were further analysed based on the size of the largest endometrioma present (Table II). For the þ 405 polymorphism, statistically significant differences in the frequencies of GG, GC and CC genotypes were observed in patients
with an endometrioma . 3 cm (4 –5 cm, P ¼ 0.04; . 6 cm, P ¼ 0.003) compared to controls. A higher frequency of GG homozygotes but not GC heterozygotes was found in cases compared to controls (Table II). However, for the 2 460C. T polymorphism, no significant differences in the frequencies of the CC, TC and TT genotypes were observed. Haplotype frequencies were estimated by the Expectation – Maximization algorithm implemented in the Alrequin 2000 software (Table III). There was strong linkage disequilibrium between the 2 460 and þ 405 alleles with a standardized disequilibrium coefficient (D0 ) of 1.000 between the two loci. The frequencies of haplotype CG, CC, TG and TC in control subjects were 44.2%, 2.4%, 28.3% and 25.0%, respectively. There was a significant deficit in the frequency of haplotype
Table I. Genotype and allele frequencies of VEGF 2460C . T and þ405G . C polymorphisms in endometriosis patients and controls Polymorphisms
2460T.C Genotype CC TC TT Allele C T þ405G.C Genotype CC GC GG Allele C G
Stage of endometriosis
Controls (210)
P-value
III (n ¼ 80)
IV (n ¼ 135)
23 (28.8) 37 (46.2) 20 (25.0)
24 (17.8) 75 (55.6) 36 (26.7)
47 (21.7) 112 (52.1) 56 (26.1)
42 (20.0) 112 (53.3) 56 (26.7)
0.89*
0.52 0.482
0.46 0.545
0.48 0.521
0.466 0.534
0.72**
1 (1.3) 26 (32.5) 53 (66.2)
3 (2.2) 45 (33.3) 87 (64.5)
4 (1.9) 71 (33.0) 140 (65.1)
18 (8.6) 79 (37.6) 113 (53.8)
0.002*
0.175 0.825
0.188 0.812
0.183 0.817
0.273 0.727
0.002**
*Fisher’s exact test (3 £ 2 table at 2 df) P , 0.05. **Fisher’s exact test (2 £ 2 table at 1 df) P , 0.05.
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Cases (215)
VEGF polymorphism and endometriosis Table II. Genotype frequencies of VEGF polymorphisms in endometriosis patients (n ¼ 198) based on size of the endometriomaa Polymorphisms
2460T.C CC TC TT P-value* þ 405G.C CC GC GG P-value*
Endometriotic cyst (cm)
Controls (210)
,3
4– 5
.6
15 (7.6)
62 (31.3)
121 (61.1)
4 (26.7) 8 (53.3) 3 (20.0) 0.76
17 (27.4) 37 (59.7) 8 (12.9) 0.08
30 (24.8) 60 (49.6) 31 (25.6) 0.25
42 (20.0) 112 (53.3) 56 (26.7)
0 (0.0) 3 (20.0) 12 (80.0) 0.1
2 (3.2) 16 (25.8) 44 (71.0) 0.04
0 (0.0) 45 (37.2) 76 (62.8) 0.003
18 (8.6) 79 (37.6) 113 (53.8)
a
All the patients studied had endometriotic cysts but the details of the size were available for only 198 patients. *Fisher’s exact test (3 £ 2 table at 2 df) P , 0.05.
TC in patients with endometriosis compared with controls (P ¼ 0.016). Discussion Association has been reported between endometriosis and a number of functional candidate genes involved in detoxification, galactose metabolism, steroid hormone production and inflammation (see up-to-date review maintained on the genetic epidemiology website http://www.well.ox.ac.uk/ , krinaz/genepi_endo.htm) (Zondervan et al., 2002b). In the present study, two SNPs in the region of the VEGF gene were investigated to ascertain whether the polymorphisms are associated with endometriosis susceptibility in South Indian women. The VEGF gene is located on chromosome 6p21.3 (Vincenti et al., 1996) and consists of eight exons exhibiting alternate splicing to form a family of proteins. Several transcription factor-binding sites are found in the VEGF 50 -untranslated region and transcriptional regulation of the gene is complex (Akiri et al., 1998). Polymorphisms within the 50 -untranslated region lead to differences in VEGF expression between individuals and could influence the etiology of a variety of pathological conditions with which VEGF has been associated. Thus, Watson et al. (2000) reported that a G allele at position þ 405, which probably lies within the myeloid zinc finger protein (MZF1) binding site, affects transcriptional activity and increases VEGF production in
peripheral blood mononuclear cells in response to lipopolysaccharide. They also showed a dose-dependent effect of the G allele: highest VEGF protein production was recorded for the GG genotype, intermediate for GC and the lowest for the CC genotype. Furthermore, the 2 460C/þ 405G haplotype has been associated with higher promoter activity, and therefore higher VEGF expression, than the 2 460T/þ 405C haplotype (Stevens et al., 2003). In the present study, the frequency of the þ 405G allele was significantly higher in women with rAFS Stage III– IV endometriosis than unaffected controls drawn from the same South Indian population, but the 2 460C. T polymorphism was not associated with the disease. Recently, Hsieh et al. (2004) showed an association between 2 460C. T polymorphism and endometriosis which is not in agreement with the present result. Possible reasons for this discrepancy could be: first, ethnic variation observed in the SNPs analysed. Indeed drastic difference in T allele distribution was observed in the two populations studied. The frequencies of mutant T allele in their controls and cases were 68.3% and 77.9% respectively compared to 53.4% and 52% in the present study (Table I). Second, the sample size in their study (cases, 122; controls, 131) is comparatively less than the sample size of the present study (cases, 215; controls, 210). We also observed that the 2 460T/þ 405C haplotype, which is associated with lower promoter activity, was significantly more common in controls than in affected women (P ¼ 0.016). The data are consistent with the previously reported finding of higher VEGF expression in women with endometriosis (Shifren et al., 1996; Donnez et al., 1998; Tan et al., 2002). Carrying the þ 405G allele may increase VEGF promoter activity leading to higher protein production and possibly a greater risk of developing the disease, whereas the þ 405C allele may be protective. VEGF transcription is regulated by estrogen in uterine endometrium (Hyder et al., 1996). Estrogens directly regulate VEGF transcription in target tissue and tumour via a complex interplay of cis- and trans-acting elements. Recently, Kawai et al. (2002) reported that BRCA-1 and the estrogen receptor a (ERa) also modulate VEGF transcription in breast cancer cells. In endometrial cells, estradiol-induced VEGF gene transcription is also estrogen receptor dependent and is activated through a variant estrogen response element (ERE) localized , 1.2 kb upstream from the VEGF transcription start site (Hyder et al., 2000). The þ 405 site is located close to the ERE and AP-1 transcription factor binding sites in
Table III. Haplotype frequencies of two VEGF biallelic polymorphismsa Haplotype frequencyb
Haplotypes 2460
405
Controls n (%)
Endometriosis n (%)
C C T T
G C G C
186 (44.3) 10 (2.4) 119 (28.3) 105 (25.0)
196 (45.6) 10 (2.3) 153 (35.6) 71 (16.5)
P-value*
Odds ratio
(95% CI)
– 0.91 0.21 0.016
Reference 0.95 1.22 0.64
0.39–2.33 0.89–1.67 0.45–0.92
a
Univariate logistic regression was used for haplotype association analysis. Observed haplotype frequencies were estimated by Expectation–Maximization method using Arlequin software v2000. *From x 2-test between control and endometriosis cases. b
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the VEGF promoter region (Stevens et al., 2003), which indicates a possible role for this polymorphism in the regulation of VEGF by estrogen. In endometriosis, neovascularization is essential for the implantation of endometrial cells in ectopic sites to be successful (Taylor et al., 2002). VEGF, a potent angiogenic factor, is involved in neovascularization, which may explain why its expression is up-regulated in the glandular epithelium of endometriosis lesions (Donnez et al., 1998). Peritoneal fluid concentrations of VEGF were significantly higher in women with rAFS Stage III– IV endometriosis (the phenotype in the present study) than in those with rAFS Stage I –II disease. The increase was attributed to the ability of endometriotic lesions to produce VEGF (Shifren et al., 1996). However, McLaren et al. (1996) demonstrated that activated macrophages are a major source of VEGF in endometriosis. They also showed that expression of this factor was regulated by steroid hormones like estradiol and progesterone. More recently, Tan et al. (2002) reported high levels of VEGF mRNA and low levels of TSP-1 (thrombospondin-1, an antiangiogenic factor) mRNA in red peritoneal endometriotic lesions. It is well established that endometriosis is an estrogendependent disease. In normal women, aromatase expression is absent in eutopic endometrium, but in endometriosis there is aberrant expression of steriodogenic factor (SF)-1, a transcription factor which increases aromatase expression and, in turn, is involved in the establishment of a positive feedback loop in favour of continuous local biosynthesis of estrogen (Kitawaki et al., 2002; Bulun et al., 2004). Aromatase promoter II activity is regulated by cyclic AMP (cAMP) and requires both CREB and SF-1 (Simpson, 2000). It is possible therefore that VEGF plays a central role in the positive feedback loop of estrogen production, and thereby in the etiology of endometriosis, by enhancing aromatase production via PI3k-Akt-CREB signalling cascade and testing of this hypothesis is going to be the focus of our future activity in this area. In conclusion we report that the þ 405G allele in the 50 -untranslated region of VEGF gene may influence the likelihood of a woman developing endometriosis.
Acknowledgements Stephen Kennedy, Mamta Deendayal and S. Shivaji would like to thank the Wellcome Trust for the funding to carry out the above work. Manjula Bhanoori would like to thank the DST, Government of India, for the WOS-A fellowship.
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