Mol. Hum. Reprod. Advance Access originally published online on December 5, 2008
Molecular Human Reproduction 2009 15(2):115-120; doi:10.1093/molehr/gan076
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Vascular endothelial growth factor genotypes and haplotypes are associated with pre-eclampsia but not with gestational hypertension
1Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Av. Bandeirantes, 3900, 14049-900 Ribeirao Preto, SP, Brazil 2Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas, 13081-970 Campinas, SP, Brazil 3Department of Gynecology and Obstetrics, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Av. Bandeirantes, 3900, 14049-900 Ribeirao Preto, SP, Brazil 4Department of Genetics, University Hospital of Medicine School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
5 Correspondence address. Tel: +55-16-3602-3163; Fax: +55-16-3633-2301; E-mail: tanus{at}fmrp.usp.br
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Abstract |
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Vascular endothelial growth factor (VEGF) is relevant for normal pregnancy, and abnormalities in VEGF functions are associated with hypertensive disorders of pregnancy. Because there are few studies on how VEGF genetic polymorphisms affect susceptibility to pre-eclampsia (PE), and no studies on how they affect susceptibility to gestational hypertension (GH), we compared VEGF genotype and haplotype distributions in normotensive and hypertensive pregnancies. Genotypes and haplotypes for VEGF polymorphisms (C-2578A, G-1154A and G-634C) were determined in 303 pregnant women (108 healthy pregnant, HP; 101 with GH and 94 with PE). When white and non-white pregnant women were considered together, no significant differences were found in the distributions of VEGF genotypes or haplotypes (P > 0.05) in the three groups. However, with only white subjects, significant differences were found in genotypes distributions for two (C-2578A and G-634C) VEGF polymorphisms (both P < 0.05) between the HP and the PE groups. Importantly, the haplotype including the variants C-2578, G-1154 and C-634, which is associated with higher VEGF gene expression, was less common in the PE group compared with the HP group (4% versus 16%; P = 0.0047). However, we found no significant differences in VEGF haplotypes distributions when the HP and GH groups were compared (P > 0.05). These findings suggest a protective effect for the ‘C-2578, G-1154 and C-634’ haplotype against the development of PE, but no major effects of VEGF gene variants on susceptibility to GH.
Key words: gestational hypertension/haplotypes/pre-eclampsia/vascular endothelial growth factor/VEGF
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Introduction |
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Pre-eclampsia (PE) is a syndrome of pregnancy characterized by hypertension and proteinuria after 20 weeks of gestation. It has been estimated that 3–5% of pregnancies are complicated by this disorder resulting in maternal and fetal complications, which include renal failure, liver failure, cerebral edema, low birthweight, prematurity and death (Walker, 2000; Sibai, 2008). In addition, it has been shown that women who develop PE have an increased risk for cardiovascular events in the future (Bellamy et al., 2007; Newstead et al., 2007). The etiology of PE remains unclear, but several alterations may have important roles in its development (Walker, 2000; Lam et al., 2005; Redman and Sargent, 2005; Palei et al., 2008; Sandrim et al., 2008). One major hypothesis is based on abnormal cytotrophoblast differentiation leading to hypoperfusion of placenta, then hypoxia and release of some soluble factors to the maternal circulation, thereby causing systemic endothelial dysfunction (Roberts et al., 1989; McCarthy et al., 1993; Zhou et al., 1997).
Vascular endothelial growth factor (VEGF) is an angiogenic factor induced by hypoxia as it initiates vasculogenesis in the placenta in coordination with other angiogenic factors (Demir et al., 2004, 2006, 2007). Importantly, PE is associated with modified cytotrophoblast expression of VEGF family ligands and receptors, and increased expression of soluble fms-like tyrosine kinase-1, which is a splice variant of VEGF receptor that captures VEGF, thus preventing its interaction with ligands and down-regulating the biological effects of VEGF (Kendall and Thomas, 1993; Kendall et al., 1996; He et al., 1999a, b; Zhou et al., 2002; Maynard et al., 2003; Ahmad and Ahmed, 2004; Chaiworapongsa et al., 2005; Smith et al., 2007). In addition to its important roles in the maintenance of endothelial integrity of many organs severely affected in PE (Esser et al., 1998), VEGF modulates the vascular tone and contributes to vascular health by suppressing endothelial apoptosis and by inhibiting leukocyte adhesion and platelet aggregation (He et al., 1999a, b; Zachary, 2001; Ferrara et al., 2003).
The relevance of VEGF for normal pregnancy and the abnormalities in VEGF functions possibly associated with PE or gestational hypertension (GH) support the idea that genetic polymorphisms in VEGF could affect the susceptibility to the development of PE or GH. Although many single nucleotide polymorphisms (SNPs) are present in the VEGF gene, three SNPs localized in promoter region have been widely studied: C-2578A, G-1154A and G-634C (Lambrechts et al., 2003; Girnita et al., 2006; Jacobs et al., 2006; Prior et al., 2006; Schneider et al., 2007). In fact, previous studies have found no associations between the C-2578A or the G-634C polymorphisms and PE (Papazoglou et al., 2004, Banyasz et al., 2006). However, it is possible that the analysis of combined effects possibly associated with these SNPs (haplotype analysis) may lead to much better information (Crawford and Nickerson, 2005; Sandrim et al., 2006a, b). In the present study, we compared the distribution of genetic variants of the three above-mentioned VEGF polymorphisms among healthy pregnant (HP), gestational hypertensives and PE women. In addition, we also examined the association of the VEGF gene haplotypes with these clinical complications of pregnancy.
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Materials and Methods |
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Subjects
Approval for the use of human subjects was obtained from the Institutional Review Board at the Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil. All patients were enrolled in the Department of Obstetrics and Gynecology, University Hospital of the Faculty of Medicine of Ribeirao Preto. We studied 303 pregnant women (108 HP women with uncomplicated pregnancies, 101 with GH and 94 with PE).
Hypertensive disorders were defined in accordance with the guidelines of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy, NHBPEP (Anonymous, 2000). GH was defined as pregnancy-induced hypertension (
140 mmHg systolic or 90 mmHg diastolic on 2 or more measurements at least 6 h apart) without significant proteinuria (<0.3 g/24 h) in a woman after 20 weeks of gestation, and returning to normal by 12 weeks post-partum. PE was defined as increased blood pressure (140 mmHg systolic or 90 mmHg diastolic on 2 or more measurements at least 6 h apart) with significant proteinuria (0.3 g/24 h) in a woman after 20 weeks of gestation.
No women with pre-existing hypertension, with or without superimposed PE, were included in the present study.
Maternal venous blood samples were collected at the time of clinic attendance. The blood collection was performed on the delivery day in HP women, and after diagnosis and initial antihypertensive treatment in hypertensive pregnant women. Leukocytes genomic DNA was extracted from 1 ml of whole blood by a salting-out method (Sambrook and Russell, 2001) and stored at –20°C until analyzed.
Genotype determination
Three clinically relevant polymorphisms in the VEGF gene were studied: C-2578A (rs699947), G-1154A (rs1570360) and G-634C (rs2010963). Genotypes were determined using the Taqman® Allele Discrimination assay (Applied Biosystems, Foster City, CA, USA). These assays use probes and primers designed by Applied Biosystems's assay-on-demand services (assay ID: C_8311602_10/-2578; C_1647379_10/-1154; C_8311614_10/-634).
TaqMan polymerase chain reaction (PCR) amplification was performed in 12 µl volumes (3 ng dried DNA, 1x TaqMan master mix, 900 nM of each primer and 200 nM of each probe) in 96-well PCR plates and fluorescence from PCR amplification was detected using a Chromo 4 Detector (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed with its software. The PCR assay was carried out following the manufacturer's instructions (Applied Biosystems) that include: one step of 10 min at 95°C (Ultra Pure AmpliTaq Gold® DNA Polymerase Enzyme Activation) followed by 40 cycles of DNA denaturation at 92°C for 15 s and annealing/extension at 60°C for 1 min.
Statistical analysis
The clinical characteristics were compared by Student's unpaired t-test. The distribution of genotypes for each polymorphism was assessed for deviation from the Hardy–Weinberg equilibrium, and differences in genotype frequency and in allele frequency between the groups were assessed using 
2 tests. A value of P < 0.05 was considered statistically significant.
The estimating haplotype (EH) software program (http://www.genemapping.cn/eh.htm, assessed on 13 October 2008) was used to estimate the haplotypes frequencies in each group. To identify which specific haplotypes are associated with increased susceptibility to PE or GH, differences in haplotype frequency were further tested using a contingency table, and value of Pc < 0.00625 (0.05/number of haplotypes 8) was considered significant to correct for the number of comparisons made. The EH program was also used to perform a linkage analysis between each pairwise combination of variants. This program calculates D' (the maximum-likelihood estimate of disequilibrium), which is a standard measure of linkage disequilibrium. The estimated disequilibrium D' values for each pairwise combination of variants were calculated as D' = D/Dmax, where D' = h– (pxq) (Marroni et al., 2005). Here, p and q are the frequencies for the rarer variants of the two polymorphisms being tested for linkage, such that p
q 0.5, and h is the frequency of the haplotype including two specific variants. When D < 0, Dmax= –pxq; when D > 0, Dmax= p (1–q). Thus, D' values can vary from +1 to –1, with a positive D' indicating that the rarer variants are associated and a negative D' indicating that the rarer variant of one polymorphism is associated with the common variant at the other locus.
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Results |
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Table I summarizes the characteristics of the 303 pregnant women enrolled in the study. HP women, women with GH and women with PE were matched by age, ethnicity, smoking, primigravida, heart rate, fasting glucose, hemoglobin and hematocrit (P > 0.05). As expected, PE and GH presented higher systolic and diastolic blood pressure compared with the HP group (Table I; both P < 0.05). Despite the criteria used to diagnose hypertensive disorders, which included systolic and diastolic blood pressure values
140 and 90 mmHg, respectively, some pregnant women had blood pressure below these diagnostic criteria at the time of blood sampling. This is because most of them were taking antihypertensive drugs. Higher body mass index was found in the GH group compared with the other study groups (Table I; P < 0.05). Lower newborn weights and gestational ages at delivery were found in the PE group compared with the other study groups (Table I; all P < 0.05). Significant proteinuria was found in PE women only (Table I).
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Table II shows the results of the single-locus analysis. The distribution of genotypes for the three polymorphisms studied here showed no deviation from Hardy–Weinberg equilibrium. Because significant interethnic differences exist in the distribution of VEGF polymorphisms (Girnita et al., 2006), we carried out two different analyses. The first analysis included white and non-white pregnant women, whereas the second one took into consideration only white pregnant women, which corresponded to 61–73% of the subjects. The first analysis showed no significant differences in genotypes and distribution of alleles for the three VEGF polymorphisms when the HP group was compared with the GH or PE groups (all P > 0.05; Table II). Conversely, when only white pregnant women were considered, significant differences were found in genotype distributions for two (C-2578A and G-634C) VEGF polymorphisms. The AA genotype for the C-2578A polymorphism and the GG genotype for the G-634C polymorphism were less common in the PE group than in the HP group (Table II; both P < 0.05).
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The linkage disequilibrium analysis was performed between each pairwise of variants including all white pregnant women (HP, GH and PE). Significant associations were found between the rarer variants -2578A and -1154A (P < 0.05; D' = +0.87), between -2578A and -634C (P < 0.05; D' = +0.66) and between -1154A and -634C (P < 0.05; D' = +0.75). The analyses of linkage disequilibrium analysis in non-white pregnant women showed similar results: D' = +0.88 (-2578A and -1154A, P < 0.05), D' = +0.84 (-2578A and -636C, P < 0.05) and D' = +0.98 (–1157A and –636C, P < 0.05).
When white and non-white pregnant women were considered, the overall haplotype frequency distributions were not significantly different in the HP group compared with the GH or the PE groups (Table III; P > 0.05). However, when taking into consideration only white women, we found significant differences in haplotype frequency distributions when the PE group was compared with the HP group (Table III; P < 0.05). Specifically, the haplotype including the variants C-2578, G-1154 and C-634 was less common in the PE group compared with the HP group (Table III; 4% versus 16%; P = 0.0047, odds ratio = 2.82, confidence interval = 0.88–9.50). No other significant differences were found in the distributions of haplotype frequencies.
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Discussion |
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This study was the first to evaluate the association of three SNPs localized in the VEGF promoter and GH or PE. Although the single locus analysis showed that two VEGF polymorphisms are associated with PE in white women, the main finding of the present study was that one VEGF haplotype ‘C-2578, G-1154 and C-634’ was less common in PE women than in HP women (odds ratio 2.82; CI 0.88–9.50). This finding suggests that this haplotype may have a protective effect against the development of PE. Conversely, our results suggest no significant effects of VEGF polymorphisms on the susceptibility to GH.
Although we found significant associations between VEGF genotypes and one VEGF haplotype with PE in white women, no significant associations were found when we took into consideration both white and non-white pregnant women together. This probably results from the fact that significant interethnic differences exist in the distribution of VEGF polymorphisms (Girnita et al., 2006), so that combining different ethnic groups obscures the genetic analysis, even though both groups were equally represented in the three experimental groups.
A few studies have examined the possible association between VEGF gene variants and PE (Papazoglou et al., 2004; Banyasz et al., 2006; Shim et al., 2007). In this regard, our findings contrast with a study showing no association between the C-2578A or the G-634C polymorphisms and PE (Papazoglou et al., 2004), although another study showed that progression of PE is affected by the C-2578A polymorphism (Banyasz et al., 2006). These discrepancies between studies may result from the lack of a precise definition of the different pregnancy-related hypertensive disorders. Indeed, the diagnostic criteria of the hypertensive disorders of pregnancy may often be equivocal. Here, we used the definitions suggested by the National High Blood Pressure Education Program Working Group on high blood pressure in pregnancy, which are widely accepted (Anonymous, 2000).
Although we found significant associations between VEGF gene variants and PE, the lack of any significant association between VEGF genotypes or haplotypes with GH is consistent with the idea that GH and PE may each have a different genetic basis, at least in terms of VEGF-related inherited components. Indeed, significant differences exist in the pathophysiological basis of these two hypertensives disorders of pregnancy, especially when the role of VEGF is taken into consideration (Simmons et al., 2000; Zhou et al., 2002; Sgambati et al., 2004). For example, although similar placental VEGF immunoreactivities were described in both healthy and gestational hypertensive pregnancies, this parameter was significantly altered in PE placentas (Sgambati et al., 2004). Moreover, differences in placental VEGF mRNA levels have been reported in GH compared with PE (Sgambati et al., 2004). These findings suggest that variable degrees of clinical severity of hypertensive disorders of pregnancy may reflect differences in placental synthesis of VEGF.
Proteinuria is a major feature and a marker of severity in PE, but not in GH. On the basis that VEGF signaling is critical for the establishment and maintenance of the glomerular filtration barrier (Eremina et al., 2007), and that anti-VEGF therapy produces proteinuria (Zhu et al., 2007), it is possible that decreased VEGF action produces the renal features of PE. In fact, dysregulation of VEGF expression within the glomerulus has been demonstrated in a wide range of renal diseases (Kang et al., 2001; Eremina et al., 2006, 2007). Therefore, it is reasonable to expect that VEGF polymorphisms would affect the development of PE, and not GH, as we have found in the present study.
Few studies have attempted to demonstrate the functional implications of VEGF genetic variants. For example, it has been shown that VEGF production by stimulated mononuclear cells from subjects with the -2578C/C or -1154G/G genotypes is significantly higher than those found in subjects with the -2578A/A and -1154A/A genotypes, respectively (Shahbazi et al., 2002). In addition, another study showed higher serum VEGF levels in subjects with the -634CC genotype when compared with subjects with other genotypes for this polymorphism (Awata et al., 2002). More interestingly, functional implications of VEGF haplotypes have also been studied. Although the present study does not address the molecular basis for the association of PE with specific VEGF haplotypes, our finding, suggesting that the haplotype ‘C-2578, G-1154 and C-634’ may have a protective effect against the development of PE, is consistent with the previous results showing that this haplotype is associated with higher VEGF gene expression than other VEGF haplotyes (Lambrechts et al., 2003; Prior et al., 2006). However, this hypothesis remains to be proved. Finally, since VEGF is coming from the placenta as well, this fetus may well contribute to changes in the maternal vasculature during pregnancy. Therefore, it is possible that the fetal genotype/haplotype may affect the susceptibility to hypertensive disorders of pregnancy.
In conclusion, we found that one VEGF haplotype (C-2578, G-1154 and C-634) was less common in PE women than in HP women, thus suggesting a protective effect for this haplotype against the development of PE. Conversely, our results suggest no significant effects of VEGF gene variants on susceptibility to GH.
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This work was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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Submitted on October 14, 2008; resubmitted on November 25, 2008; accepted on December 1, 2008.
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