Vascular endothelial growth factor gene polymorphisms in North Indian patients with recurrent miscarriages

Article Outline

• Abstract
• Introduction
• Materials and methods
  • VEGF genotyping
  • Statistical analysis
• Results
• Discussion
• Acknowledgements
• Appendix A. Supplementary data
• References
• Copyright

Abstract 

The association of four common polymorphisms of vascular endothelial growth factors (VEGF) with recurrent miscarriages (RM) was evaluated in North Indian women for 200 patients with RM and 200 controls. The subjects were genotyped for the polymorphisms −2578C/A, −2549 18-bp I/D, −1154G/A and +936C/T. Association of VEGF genotypes, alleles and haplotypes with recurrent miscarriage were evaluated by Fisher’s exact test. −1154G/A and +936C/T modified the risk of RM. The −1154A allel and +936T allel significantly increased the risk of RM (OR=1.485, P=0.0210, 95% CI 1.072–2.057 and OR=1.869, P=0.0054, 95% CI 1.214–2.876 respectively). Risk was further increased when –1154A/A genotype and +936C/T genotype were considered (OR=2.0, P=0.0310, 95% CI 1.068–3.747 and OR=1.716, P=0.0293, 95% CI 1.058–2.784 respectively). However, no association was found between −2578C/A or −2549 18-bp I/D and RM. Four haplotypes, AIAC, ADAC, CIAT and ADGT, were found to predispose to RM while the haplotypes CIAC, CDGT and ADGC were found to show protective effect. In conclusion, two common polymorphisms of the VEGF gene, −1154G/A and +936C/T, increase the risk of RM in North Indian women. RM is also predisposed in the presence of haplotypes AIAC, ADAC, CIAT and ADGT.

In many of the women with three or more miscarriages, no definite cause for their illness can be found. Recently researchers have found that a growth factor which promotes the growth of blood vessels known as vascular endothelial growth factor (VEGF) plays an important role in the formation of blood supply of the fetus through the placenta. Reduced concentrations of this factor might contribute to the improper formation of the placenta, which may cause recurrent miscarriage. The gene for VEGF is polymorphic consisting of variations in its nucleotide sequence at various locations. Researchers have shown that these variations in the gene lead to different concentrations of VEGF in different people. Hence, there may be some women with a certain polymorphism(s) who have lower VEGF production and so are at risk of recurrent miscarriage. In this study conducted in North India, we tested these polymorphisms in women with and without recurrent miscarriage and tried to find if any particular polymorphism increased the probability of a woman having a recurrent miscarriage. We found that at least two polymorphisms of the VEGF gene, −1154 G/A and +936C/T, did increase the risk of recurrent miscarriage among carriers of the uncommon allele.

Keywords: angiogenic factors, miscarriage, polymorphism, uteroplacental insufficiency, vascular endothelial growth factor (VEGF)

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Introduction 

Recurrent miscarriage (RM), i.e. the occurrence of three consecutive spontaneous miscarriages, afflicts 0.5–3.0% women. Uteroplacental insufficiency, which is important in the pathogenesis of pre-eclampsia and intrauterine growth restriction, is believed to be responsible for some of these cases of RM (Norwitz, 2006).

Vascular endothelial growth factor (VEGF) plays an important role in the establishment of uteroplacental circulation. This cytokine is a potent mitogen and helps in the survival of endothelial cells. It participates in placentation by increasing angiogenesis, vascular permeability and trophoblast invasion, which are required for successful implantation (Ferrara et al., 2003, Nardo, 2005). The VEGF gene is highly polymorphic. The common polymorphisms reported by various studies are: in the promoter region, −2578C/A, −1154G/A, −2549 18-bp I/D, −460C/T, −1498T/C, −1190G/A; in the 5′ untranslated region, −634G/C, −7T/C; and in the 3′ untranslated region, +936C/T and +1612G/A (Awata et al., 2002, Brogan et al., 1999, Shahbazi et al., 2002). The frequency of these polymorphisms is varied across different populations (Awata et al., 2002, Buraczynska et al., 2007, Coulam and Jeyendran, 2008, Lee et al., 2009, Papazoglou et al., 2004, Papazoglou et al., 2005, Prior et al., 2006, Salvarani et al., 2004, Shahbazi et al., 2002, Uthra et al., 2008). The −1154G/A, −2578C/A, −2549I/D and +936C/T polymorphisms have been demonstrated to be associated with differential VEGF production in various studies (Brogan et al., 1999, Renner et al., 2000, Salvarani et al., 2004, Shahbazi et al., 2002). Various diseases have been reported to be associated with these polymorphisms and the hypothesis proposed is the involvement of VEGF in angiogenesis. As RMs are associated with uteroplacental insufficiency, it is proposed that women carrying the low expression alleles may be predisposed to impaired placentation, which may result into RM.

Prior to this study, there have been reports from a few groups evaluating the association of VEGF polymorphisms with RM (Coulam and Jeyendran, 2008, Goodman et al., in press, Goodman et al., 2008, Lee et al., 2009, Papazoglou et al., 2005). All these studies have reported an increased prevalence of the −1154A allele in women with RM. In contrast, in both the studies where the −2578, −634 and +936 polymorphisms were evaluated, no association of these variations with RM was found (Lee et al., 2009, Papazoglou et al., 2005). The present study evaluated the association of −2578C/A, −1154G/A, −2549 18-bp I/D, −and +936C/T polymorphisms and their haplotypes with RMs in the Indian population. This is the first study from India evaluating these polymorphisms in RM patients.

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Materials and methods 

This case control study included 200 patients (mean age 28±5.4years) and 200 controls (31.9±7.3years) from the outpatient clinic of the Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences and Queen Mary Hospital CSSMU, two tertiary care institutions situated in Lucknow, a city in the Northern part of India. The inclusion criteria for the patients were women with three or more consecutive miscarriages before 20weeks of gestation. All patients were investigated for anatomic uterine defects, immunological causes, chromosomal abnormalities and hormonal imbalances. Factor V Leiden mutation analysis was also performed for all patients. All patients with abnormalities found in any of these tests were excluded from the study. Only patients with unexplained miscarriages were included in the study. Controls were women with at least two live births and no previous history of miscarriages, pre-eclampsia or ectopic pregnancy. Informed consent was taken from all patients and controls after providing them with an information sheet. The study was approved by the institutional ethics committees of the study centres. For all the patients and controls, DNA was extracted from venous blood using QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, US).

VEGF genotyping 

Genotyping was performed for −2578C/A and −1154G/A polymorphisms using allele refractory mutation system PCR and for +936C/T polymorphisms using restriction fragment length polymorphism as described by Papazoglou et al. (2005). The −2549I/D polymorphism was genotyped using a common set of primers as mentioned by Buraczynska et al. (2007). The PCR and restriction fragment length polymorphism products were run on 2% agarose and visualized using ethidium bromide.

Statistical analysis 

Differences in the VEGF genotype, allele frequencies and haplotypes between the study and control groups were analysed with the Fisher’s exact test. P-values <0.05 were considered statistically significant. The odds ratio (OR) was used as a measure of the strength of association between genotypes, allele frequencies and haplotypes with RM. Analysis for the genotypes was done under additive and recessive, as well as dominant models of inheritance. Haplotypes were generated using Arlequin software and statistical analysis was performed using the Statistical Package for Social Sciences version 13 (SPSS, USA). Fisher’s exact test was used to calculate OR and P-value <0.05 was taken as significant.

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Results 

The genotype frequencies of the four polymorphisms are shown in Table 1. The mutant homozygous genotype (AA) of the −1154G/A polymorphism was significantly more prevalent in the cases as compared with controls and increased the risk of RM two times (OR 2.00, P=0.0310). The heterozygous genotype (GA) was also more prevalent in the RM group but was statistically insignificant. The recessive model also showed increased risk of RM (OR 1.926, P=0.0485). The variant (CA and AA) genotypes of −2578C/A polymorphism were more common in the cases as compared with controls; however, this was not statistically significant for any of the three models. For the +936C/T polymorphism, the heterozygous CT genotype was significantly associated with RM (OR 1.716, P=0.0293). The dominant model of inheritance also showed significant association of the variant +936 genotypes (CT+TT) with RM (OR 1.861, P=0.013). The TT genotype was found in six cases and only one control. The −2549I/D polymorphism genotypes did not show significant association with RM under any of the three inheritance models.

Table 1. Genotypes of vascular endothelial growth factor polymorphisms in cases and controls.

Genotype	Cases (n=200)	Controls (n=200)	OR	95% CI	P-value
1154
AA (additive model)	32 (16.0)	18 (9.0)	2.000	1.068–3.747	0.0310
GA (additive model)	48 (24.0)	47 (23.5)	1.149	0.7170–1.841	NS
GG	120 (60.0)	135 (67.5)	1.000
AA versus GA+GG (recessive model)			1.926	1.042–3.561	0.0485
AA+GA versus GG (dominant model)			1.385	0.9194–2.085	NS
2578
AA (additive model)	23 (11.5)	17 (8.5)	1.524	0.7712–3.010	NS
CA (additive model)	74 (37.0)	67 (33.5)	1.244	0.8141–1.901	NS
CC	103 (51.5)	116 (58.0)	1.000
AA versus CA+CC (recessive model)			1.399	0.7228–2.707	NS
AA+CA versus CC (dominant model)			1.301	0.8764–1.930	NS
936
TT (additive model)	6 (3.0)	1 (0.5)	–	–	–
CT (additive model)	52 (26.0)	35 (17.5)	1.716	1.058–2.784	0.0293
CC	142 (71.0)	164 (82.0)	1.000
TT+CT versus CC (dominant model)			1.861	1.160–2.985	0.0130
2549
DD (additive model)	45 (22.5)	46 (23.0)	0.9130	0.5272–1.581	NS
ID (additive model)	95 (47.5)	98 (49.0)	0.9048	0.5707–1.434	NS
II	60 (30.0)	56 (28.0)	1.000
DD versus ID+II (recessive model)			0.9719	0.6089–1.551	NS
DD+ID versus II (dominant model)			0.9074	0.5890–1.398	NS

Values are number (%).

Additive model=comparing mutant homozygous and heterozygous genotypes individually with wild homozygous genotypes; CI=confidence Interval; dominant model=mutant homozygous and heterozygous genotype taken together compared with wild homozygous genotype; NS=not statistically significant; OR=odds ratio; recessive model=comparing mutant homozygous genotype with wild homozygous and heterozygous genotype taken together.

The allele frequencies of the four polymorphisms are shown in Table 2. The −1154A allele was significantly more prevalent in the cases with OR 1.485 (P=0.0210). The association of +936T allele with RM was found to be highly significant (OR 1.869, P=0.005).

Table 2. Allele frequencies of vascular endothelial growth factor polymorphisms in cases and controls.

Values are number (%).

CI=confidence interval; NS=not statistically significant; OR=odds ratio.

The haplotype analysis revealed the presence of 16 and 14 haplotypes in the cases and controls, respectively (Supplementary Table 1, available online only). Two haplotypes, ADAT and AIGT, were found only in cases and not in controls. The more prevalent haplotypes and the ones which had protective or risk predisposing effect are shown in Table 3. AIAC, ADAC, CIAT and ADGT were predisposing haplotypes. Protective effect was observed for the haplotypes CIAC, CDGT and ADGC.

Table 3. Haplotypes of vascular endothelial growth factor in cases and controls.

Haplotypes generated using Arlequin software. Statistical significance was analysed using Fisher’s exact test.

CI=confidence interval; OR=odds ratio; NS=not statistically significant; VEGF=vascular endothelial growth factor; RM=recurrent miscarriage.

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Discussion 

Uteroplacental insufficiency is believed to be responsible for some cases of unexplained recurrent miscarriages (Norwitz, 2006). The establishment of uteroplacental circulation involves three cardinal steps: (i) adequate uterine angiogenesis/vascularity at the time of implantation; (ii) development and expansion of the villous vasculature soon after implantation; and (iii) remodelling of the maternal uterine circulation near the maternal–fetal interface (Torry et al., 2004). VEGF plays an important role in this process by participating in the increased angiogenesis, enhancing vascular permeability and trophoblast invasion necessary for successful implantation (Nardo, 2005). A significant rise in circulating concentrations of free and total VEGF during early pregnancy has been seen (Molskness et al., 2004). Mechanisms responsible for the decreased angiogenesis in recurrent pregnancy losses may be related to decreased expression of VEGF and its receptors which has indeed been demonstrated in decidual and villous tissue from aborted pregnancies (Vuorela et al., 2000).

VEGF, also referred to as VEGF-A, is a major regulator of normal and abnormal angiogenesis. It is a potent mitogen and survival factor for endothelial cells. It also increases vascular permeability and mediates vasodilatation by nitric oxide release. There are many isoforms of VEGF but VEGF165 is the predominant physiologically active form. It acts by binding to two tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (also known as kinase domain region (KDR) or Flk-1). VEGFR2 is the key signalling receptor through which VEGF mediates its angiogenic action (Ferrara et al., 2003).

The VEGF gene is highly polymorphic and there are variations in the distribution of these polymorphisms in different populations and also in various disease states. Many of its polymorphisms are known to affect VEGF plasma concentrations presumably by altering gene transcription. To evaluate the effect of VEGF polymorphisms on VEGF plasma concentrations, many functional studies have been attempted ((Brogan et al., 1999, Renner et al., 2000, Salvarani et al., 2004, Shahbazi et al., 2002). These studies demonstrate that mutant alleles of −1154G/A, −2578C/A, −2549I/D and +936C/T lead to low VEGF expression. Although interplay of multiple factors contributes to the aetiology of RM, it is proposed that the low expression alleles of VEGF may predispose to RM by leading to impaired placentation. The current study investigated the frequency of these polymorphisms in North Indian women with and without RM (as controls).

Literature reports show wide variability in the genotype and allele frequencies of the VEGF gene in different populations. The −1154A allele is found in 6% of Blacks (Prior et al., 2006), 18% of Orientals (Lee et al., 2009) and 31% of Europeans (Papazoglou et al., 2005, Shahbazi et al., 2002). On the other hand the −2578A allele is present in 14% Blacks (Prior et al., 2006), 27% Orientals (Lee et al., 2009) and 46–52% Europeans (Papazoglou et al., 2004, Papazoglou et al., 2005, Shahbazi et al., 2002). The 936T allele occurs with a frequency of 10–12% in Europeans (Salvarani et al., 2004, Papazoglou et al., 2004, Papazoglou et al., 2005) and 15% in Asians (Lee et al., 2009, Uthra et al., 2008). Homozygosity for this allele is rare, occurring in 0.8–4% of people across different populations. It was not found in even one patient from a cohort of 212 diabetic South Indians in one of the Indian studies (Uthra et al., 2008) that reported the frequency of T allele as 9%. The 2549D allele is reported to occur in 50–60% of Europeans and DD genotype is seen in 22–42% within this population group (Buraczynska et al., 2007, Salvarani et al., 2004).

The current study found −1154A/A genotype in 9.0%, −2578A/A in 8.5%, −2549D/D in 23.0% and +936T/T in 0.5% among healthy parous women. The frequencies of the corresponding mutant alleles in controls was found to be 21%, 25%, 48% and 9% for the 1154A, 2578A, 2549D and 936T allele, respectively. As there is no prior data for comparison on all the four polymorphic sites studied, a comparative analysis could not be performed. Only the +936T allele and the corresponding homozygous genotype have been studied in a report from South India and the current results are in concordance with this study (Uthra et al., 2008).

The various polymorphisms of the VEGF gene have been shown to predispose to various disease states. The +936 polymorphism has been associated with pre-eclampsia (Papazoglou et al., 2004). Two previous studies, one from the Greek population (Papazoglou et al., 2005) and the other from the Korean population (Lee et al., 2009), have not found any effect of this polymorphism on the risk of RM. However, the current study found that the mutant T allele significantly predisposes to RM. The CT genotype (under additive model) and the CT and TT genotypes (under dominant model) are found to be positively associated with RM. The 936T allele carriers have been shown in previous reports to have low plasma VEGF concentrations (Renner et al., 2000). This is presumed to occur due to reduced transcription of the gene as a result of reduced binding of transcription factor AP-4 to the polymorphic site (Renner et al., 2000). Hence, the association of RM with T allele carriers appears biologically plausible.

The −1154G/A polymorphism in the promoter region is also believed to regulate gene expression, with the A allele leading to reduced plasma VEGF concentrations (Brogan et al., 1999, Shahbazi et al., 2002). In previous studies, the A allele has been demonstrated to increase the risk of recurrent miscarriage by approximately two-fold. (Coulam and Jeyendran, 2008, Goodman et al., in press, Goodman et al., 2008, Lee et al., 2009, Papazoglou et al., 2005). In the present study, it was also found that carriers of A allele are at about 1.5 times higher risk of undergoing recurrent pregnancy loss. The genotype analysis for this polymorphism has also shown a statistically significant two-times increased risk of RM in carriers of AA genotype.

The −2578C/A (Brogan et al., 1999, Shahbazi et al., 2002) and −2549I/D (Brogan et al., 1999, Salvarani et al., 2004) polymorphisms are also known to be associated with differential gene expression. The −2578 polymorphism was not found to be associated with RM in two previous studies (Lee et al., 2009, Papazoglou et al., 2005). The −2549 polymorphism has never been studied in RM and ours is the first study for −2549 polymorphism; however, no association was found with the patient or control groups.

The haplotype analysis has revealed that the four haplotypes, AIAC, ADAC, CIAT and ADGT are predisposing haplotypes. Interestingly two haplotypes, ADAT and AIGT, although found in a few patients, were absent in our control group population. Three haplotypes, CIAC, CDGT and ADGC, were found to have protective effect. In a previous study from South Korea (Lee et al., 2009), haplotype analysis for four polymorphisms, −2578C/A, −1154G/A, −634G/C and +936C/T, showed that haplotype AAGT was significantly associated with RM and that the haplotypes CAGT and AAGT also marginally increased the risk of RM. No other studies involving haplotype analysis of VEGF in RM are available in the literature. VEGF haplotypes have previously been correlated with VEGF plasma concentrations (Prior et al., 2006). Particular haplotypes especially those involving low expression alleles may predispose to RM due to decreased VEGF production.

To conclude, the current study has reinforced the findings of the previous reports by finding increased risk of RM in carriers of −1154A allele and AA genotype. It has been found that there is a significant increased risk of RM in women with +936T allele, both at the allele and genotype levels. The association of VEGF polymorphism with pre-eclampsia, the other disorder of defective placentation is already known. Trials of VEGF therapy in animal models of pre-eclampsia have shown positive results (Li et al., 2007). Further research may reveal therapeutic benefit of VEGF therapy in women with unexplained recurrent miscarriages.

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Acknowledgements 

The grant for this project was kindly provided by the Department of Science and Technology, Government of India, New Delhi, India.

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Appendix A. Supplementary data 

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