−765 G→C and −1195 A→G Promoter Variants of the Cyclooxygenase-2 Gene Decrease the Risk for Preeclampsia

Abstract

Cyclooxygenase-2 (COX-2) is the inducible isoenzyme of COX that leads to increased production of prostaglandins and thromboxane, the mediators of inflammation. Controversial data regarding COX levels or activities in the placentas of women with preeclampsia have led us to examine whether a single nucleotide polymorphism (SNP) in the COX-2 gene is associated with the onset of preeclampsia. Two polymorphisms in the promoter region of COX-2 gene were examined by the polymerase chain reaction and restriction fragment length polymorphism in 128 controls and 74 preeclamptic patients. Genotype distribution and allelic frequencies for −765G→C polymorphism of COX-2 gene were significantly different between patients and controls (p=0.000 and p=0.042, respectively). The odds ratio (OR) for preeclampsia risk associated to the −765G allelic variant was 4.07 (95% confidence interval [CI]: 0.89-18.56). The AA genotype of the −1195 A→G variant was present at a significantly higher frequency among all preeclamptic subjects (p=0.000 χ²: 13.4, OR: 3.44, 95% CI: 1.74-6.77). A moderate linkage was observed between the −765G and −1195A variants (D⁰: 0.201; r²: 0.003). These findings suggest that SNPs, −765G→C and −1195 A→G, on the promoter region of COX-2 gene may reduce the risk of preeclampsia, possibly by affecting the rate of gene expression.

Introduction

Cyclooxygenase (COX) is the key enzyme in the biosynthesis of prostaglandins and thromboxane from arachidonic acid. Two isoenzymes, COX-1 and COX-2 have been identified, the former as a regulator of physiologic functions and the latter as a mediator in pathophysiologic reactions such as inflammation. COX-2 (prostaglandin synthase 2) is the inducible form of COX that leads to increased production of prostaglandins and thromboxane, the mediators of inflammatory processes. Thromboxane is also a potent vasoconstrictor and can contribute to endothelial dysfunction (Félétou et al., 2011).

Preeclampsia is defined by the presence of hypertension and proteinuria during pregnancy. Enhanced production of vasoconstrictor metabolites leading to hypertension is the main characteristic of this syndrome. The expression of COX-2 in systemic vasculature of women with preeclampsia has been reported (Shah and Walsh, 2007). COX-2 protein and mRNA expression take place in fetal membranes and placenta beginning from the second trimester with a marked increase after the 30th week of normal pregnancy (Slater et al., 1999). Placental levels of COX-1 and/or COX-2 are increased in healthy pregnancy at term (Hirst et al., 1998; Hirsch et al., 2005). In preeclamptic subjects, COX-2 is expressed in umbilical vein endothelial cells and in trophoblasts too (Woodworth et al., 1994; Johnson et al., 1995; Akarasereenont et al., 1999). In addition, significantly more expression of this enzyme was observed in neutrophils, and the release of COX-2 inflammatory products that infiltrated the vasculature was thought to be responsible for vascular dysfunction and vasoconstriction (Bachawaty et al., 2010). However, reports are controversial regarding COX levels or activities in preeclamptic pregnancies. Previous studies revealed that COX-2 levels in the placenta of preeclamptic women are reduced (Khan et al., 1999), and activities of COX-1 and COX-2 are decreased (Börekçi et al., 2006). Despite the studies indicating significant alterations in the cyclooxygenase expression, it has been reported that COX-1 and COX-2 mRNAs and protein expression in placenta remain unchanged in preeclamptic pregnancies (Wetzka et al., 1997).

A number of single nucleotide polymorphisms (SNPs) in the promoter region of the COX-2 gene, which are associated with altered transcriptional activity, have been described. A novel promoter polymorphism in the COX-2 gene (−765G→C) that reduces promoter activity by 30% compared with the wild type allele has been demonstrated (Papafili et al., 2002). Polymorphisms on the COX-2 gene that might modify the levels of protein expression would be anticipated to have a substantial influence on disease phenotype. Therefore, we sought to understand the role of two COX-2 polymorphisms (−1195A→G and −765G→C) in the onset of preeclampsia.

Subjects and Methods

Study groups

Seventy-four pregnant women with preeclampsia who had a diastolic blood pressure higher than 110 mmHg and systolic blood pressure higher than 140 mmHg were included in the study. Healthy controls consisted of normotensive women who had previously undergone ≥2 term pregnancies unaffected by preeclampsia (n=128). Preeclampsia was defined as a condition in pregnancy when blood pressure ≥140/90 mmHg is noted on two different occasions >4 h apart in a previously normotensive woman, associated with proteinuria in excess of 300 mg/L in a 24 h collection. All the subjects enrolled in the study were Caucasians. They had a similar socioeconomic status and were applied to Bakirkoy State Hospital, Division of Obstetrics or Istanbul Faculty of Medicine, Department of Obstetrics and Gynecology. All participants gave an informed consent prior to study and the Local Ethical Committee approved the study protocol. The procedures were carried out in accordance with The Code of Ethics of the Declaration of Helsinki.

The diagnosis of preeclampsia was confirmed by the clinical and laboratory examinations. Protein was measured in 24-h urine samples using a Cobas Integra 400 plus autoanalyzer, Roche.

Polymorphism analysis

Blood samples from all study participants were collected in EDTA-containing tubes. Genomic DNA was extracted from peripheral whole blood according to the salting-out technique (Miller et al., 1988). Genotyping was performed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP); the procedures of PCR-RFLP are given in Table 1 (Shi et al., 2004; Zhang et al., 2005). The PCR amplifications were performed using the GeneAmp PCR Systems 9700 (Applied Biosystem). The appropriate primers were used to amplify the corresponding gene of the subjects by PCR and the reaction products were digested by using the appropriate enzyme at 37°C. The digested products were analyzed on 2% agarose gel stained with ethidium bromide and examined under transillumination. Each gel was read by two observers unaware of the subject's status. If there is any conflict, genotyping of samples was repeated. The expected results after restriction for each gene were also given in Table 1.

Table 1.

Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Procedures and Products of −765G→C and −1195A→G Genotypes of Cyclooxygenase-2

COX-2	Primers (forward and reverse)	PCR product	Restriction enzyme	Restriction products
−765G→C (rs20417) (2)	5′-AGG CAG GAA ACT TTA TAT TGG-3′ 5′-ATG TTT TAG TGA CGA CGC TTA-3′	309 bp	AciI	CC: 309 bp GG: 209 bp, 100 bp GC: 309 bp, 209 bp, 100 bp
−1195A→G (rs689466) (3)	5′-CCCTGAGCACTACCCATGAT-3′ 5′-GCCTTCATAGGAGATACTGG-3′	173 bp	PvuII	AA: 273 bp GG: 220 bp, 53 bp AG: 273 bp, 220 bp, 53 bp

PCR, polymerase chain reaction.

Statistical analysis

Statistics were performed using the SPSS software package (revision 11.5 SPSS Inc., Chicago, IL). Data are expressed as means±SD. Differences in the distribution of −765G→C and −1195A→G genotypes or alleles between cases and controls were tested using the chi-square (χ²) test. Allelic frequencies were calculated by gene counting. Fisher's exact test was used if the number in any cell of the 2×2 contingency table was <5. Relative risk at 95% confidence intervals (CI) was calculated as the odds ratio (OR).

Linkage between COX-2 −765G→C and −1195A→G polymorphisms was assessed using D⁰ and r² values obtained through the Haploview Program (www.broad.mit.edu/mpg/haploview/documentation.php). p-values<0.05 were considered statistically significant. The NCSS 2000 statistical package was used to evaluate the power analysis.

Results

The frequencies of −765G→C and −1195A→G genotypes among patients and controls are shown in Table 2. The frequency of COX-2 −765GG genotype was higher in preeclamptics than in controls (67.6% vs. 40.6%). Genotype distribution and allelic frequencies for the −765G→C polymorphism of COX-2 gene were significantly different between patients and controls (p=0.000). Individuals who had GG genotype showed an increased risk of preeclampsia (p=0.000 by Fisher's exact test, χ²:13.6, OR=3.04, 95% CI=1.66-5.55). G allele frequency in the patient group was higher than in the control group (82.4% vs. 65.2%, p=0.000, Table 3). The OR for preeclampsia risk associated to the −765G allelic variant was 4.07 (95% CI: 0.89-18.56, p=0.042 by Fisher's exact test).

Table 2.

The Frequencies of −765G→C and −1195A→G Variants of Cyclooxygenase-2 in Patients and Controls

	Controls		Patients
Genotypes/alleles	n:128	(%)	n:74	(%)	p
−765 G→C
GG	52	(40.6)	50	(67.6)
CC	13	(10.2)	2	(2.7)
CG	63	(49.2)	22	(29.7)	^a0.000 χ²:14.4
G allele	167	(65.2)	122	(82.4)
C allele	89	(34.7)	26	(17.5)	^b0.000 χ²:13.6
−1195 A→G
AA	71	(55.5)	60	(81.1)
GG	-		-
AG	57	(44.5)	14	(18.9)	^a0.000 χ²:13.4
A allele	199	(77.7)	134	(90.5)
G allele	57	(22.2)	14	(9.4)	^b0.001 χ²:10.6

Numbers in parentheses indicate percentages.

For the genotype distribution between the groups.

For the allelic frequency.

Table 3.

Haplotype Associations of −765G→C and −1195A→G Polymorphisms

		Frequency
Haplotype number	Haplotype associations	Overall	Patients	Controls	χ²	p
1	−765G: −1195A	0.580	0.741	0.486
2	−765C: −1195A	0.245	0.165	0.291	8.106	0.004
3	−765G: −1195G	0.136	0.083	0.166	5.436	0.019
4	−765C: −1195G	0.040	0.011	0.057	5.077	0.024

The AA genotype of the −1195 variant on COX-2 was present at a significantly higher frequency among preeclamptic subjects, over 80% of the preeclamptic individuals being −1195A homozygotes compared with 55% of the control subjects (p=0.000 χ²: 13.4, OR: 3.44, 95% CI: 1.74-6.77).

The −1195 G allele frequency was significantly less in preeclamptics than in controls (9.45% vs. 22.2%; p=0.001, χ²:10.6, Table 2). A moderate linkage was observed between −765G→C and −1195A→G polymorphisms (D⁰: 0.201, r²: 0.003).

In addition to genotype and alleles, we evaluated the haplotype frequencies. The frequency of COX-2 −765G: −1195A haplotype in the patient group was significantly higher than in controls (Table 3).

Discussion

This study was focused on the possible association of two SNPs on the promoter region of COX-2 gene with preeclampsia in a Turkish population. It has been shown that the −765G→C polymorphism on COX-2 gene confers protection against cardiovascular diseases and colorectal adenoma in humans (Cipollone et al., 2004; Ulrich et al., 2005). In a recent study carried out in our laboratory, it was found that the presence of the polymorphic −765C variant also decreases the risk of coronary artery disease in a Turkish population (Ol et al., 2011). Accordingly, we observed a significant negative association between the polymorphic −765C and −1195G variants of the COX-2 gene and the occurrence of preeclampsia during pregnancy. In preeclampsia, thromboxane production rate is increased. In a recent study, it was found that preeclamptic pregnants excreted significantly elevated thromboxane metabolites compared to healthy pregnants (Perneby et al., 2011). COX-2 activity is one of the rate-limiting steps in prostaglandin/thromboxane synthesis. Since the −765C variant of the COX-2 gene has relatively lower transcription rate, the synthesis of prostaglandins and tromboxane is expected to be less in subjects bearing the polymorphic phenotype, thus ameliorating the state of inflammation. It has been reported that the −765C variant is associated with low levels of C-reactive protein; therefore it may be protective in cardiovascular disease, although it may facilitate infection in certain conditions (Papafili et al., 2002). In newborns, carriage of the COX-2 C allele has been found negatively associated with lesions of placental ischemia/malperfusion (Polydorides et al., 2007). However, the incidence of preeclampsia in pregnant women carrying either C allele for the −765G→C variant or G allele for the −1195A→G variant has not been previously evaluated. Our study is the first to show that polymorphic variants of COX-2 gene may be significantly effective in reducing the risk of preeclampsia. In pregnant women bearing polymorphic alleles, changes in biochemical parameters related to inflammation and/or hypertension might be evaluated in further investigations to elucidate the role of COX-2 activity in the pathogenesis of preeclampsia.

Footnotes

Disclosure Statement

No competing financial interests exist.

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