Genetic analysis of eNOS gene polymorphisms in association with recurrent miscarriage among North Indian women

Abstract

This study investigated the association of common polymorphisms of the endothelial nitric oxide synthase (eNOS) gene with recurrent miscarriage (RM) among North Indian women. A total of 200 patients with unexplained recurrent miscarriages and 300 controls were genotyped for six polymorphic regions of eNOS by PCR, re-sequencing and RFLP. The GG genotype of 12862A>G, the G allele of Glu298Asp and the aa genotype of intron 4VNTR increased the risk of RM by ∼1.8-fold, ∼3.5-fold and ∼2-fold, respectively (odds ratio (OR) 1.84, 95% confidence intervals (CI) 1.19–2.86, P = 0.0066; OR 3.58, 95% CI 2.12–6.03, P < 0.0001; and OR 2.23, 95% CI 1.04–4.77, P = 0.0493). Two haplotypes were found to have a significant protective effect against RM (OR 0.63, 95% CI 0.48–0.82, P = 0.0009; and OR 0.4, 95% CI 0.19–0.81, P = 0.0149) and another was found to increase the risk of RM by ∼2-fold (OR 2.12, 95% CI 1.16–3.89 P = 0.0195). In conclusion three common polymorphisms of eNOS gene, 12862A>G, Glu298Asp and intron 4VNTR increase the risk of RM in North Indian women. Risk of RM may also be modified by the presence of particular haplotypes.

In many women who have had three or more miscarriages, no definite cause for the condition can be found. It was recently found that implantation depends on the receptivity of maternal endometrium which in turn is influenced by synergistic action of nitric oxide (NO). The production of NO in endothelial cells increases during pregnancy and contributes to vasodilation and vasopressor responses. NO is synthesized by a family of NO synthase (NOS) enzymes in which three isoforms have been identified: neuronal (nNOS, NOS-1), inducible (iNOS, NOS-2) and endothelial (eNOS, NOS-3). It has been shown that polymorphisms in the coding and non-coding regions of eNOS may alter eNOS expression and/or activity and thus cause a decrease in NO synthesis which may predispose patients to hypertension, vasospasm and atherosclerosis, renal failure, pre-eclampsia and recurrent miscarriages. In this study conducted in North India, we tested polymorphisms of the eNOS gene in women with and without recurrent miscarriage and tried to find if any particular polymorphism increased the probability of a woman having recurrent miscarriage. We found that at least three common polymorphisms of the eNOS gene – 12862A>G, Glu298Asp and intron4 VNTR – increase the risk of recurrent miscarriage in North Indian women.

Introduction

Recurrent early pregnancy loss or recurrent miscarriage (RM) is one of the most common reproductive problems and remains one of the least understood pathological conditions. It is defined as three or more consecutive losses before the 20th week of gestation, affecting 0.5–2% of pregnant women. The establishment of a successful pregnancy is a complex process, where the events taking place during early to mid gestation, from fertilization, implantation of blastocyst, differentiation of trophoblast to invasion of the endometrium by the trophoblast along with establishment of the feto–maternal interface, are brought about by careful regulation of the interplay of multiple factors which either infiltrate or are expressed in the uterine microenvironment (Hanna et al., 2006). Implantation depends on the receptivity of maternal endometrium, which is influenced by synergistic action of nitric oxide (NO). NO is generated by endothelium and is a potent vasodilator and acts as an important key factor in the anti-atherosclerotic properties of the endothelium. It takes part in a variety of physiological functions in the human body including vasodilation, inhibition of platelet aggregation and vascular smooth muscle relaxation (Ignarro et al., 1987). NO may also be directly involved in the physiology of reproductive organs and is implicated in the development of endothelial damage, hypertension, coronary spasm and myocardial infarction (Hingorani, 2003). The production of NO in endothelial cells increases during pregnancy and contributes to vasodilation and vasopressor response. NO is synthesized during the conversion of amino acid l-arginine to l-citrulline by a family of NO synthase (NOS) enzymes in which three isoforms have been identified (Lapointe et al., 2006): neuronal (nNOS, NOS-1), inducible (iNOS, NOS-2) and endothelial (eNOS, NOS-3). Endothelial nitric oxide synthase (eNOS or NOS-3) is the main contributor of circulating NO.

The gene for eNOS is located on chromosome 7 (7q35-q36), as a single copy of 26 exons with an entire length of 21 kb, and encodes an mRNA of 4052 nucleotides (Marsden et al., 1993). Disruption of the eNOS gene leads to hypertension in mice (Van Vliet et al., 2007) and inhibition elevates blood pressure in healthy humans. eNOS is a constitutively expressed isoform whose activity is dependent on the intracellular changes in Ca²⁺ concentrations. eNOS expressed in the terminal villous vessels in the syncytiotrophoblast is the main enzyme required for vascular NO production and influences placental human chorionic gonadotrophin production during gestation. Trophoblast cells in the first trimester express high amounts of eNOS (Al-Hijji et al., 2003). Studies undertaken to identify eNOS gene variations have revealed that many cis- and trans-acting factors regulate the expression of eNOS and that its expression level in turn corresponds directly to the amount of NO in the blood (Karantzoulis-Fegaras et al., 1999). It has been shown that polymorphisms in the coding and non-coding regions of eNOS may alter eNOS expression and/or activity and thus cause a decrease in NO synthesis (Logan et al., 2005), which may predispose patients to hypertension, vasospasm and atherosclerosis, renal failure, pre-eclampsia and RM (Akcay et al., 2004, Fatini et al., 2006, Wang and Wang, 2000, Xin et al., 2009).

Genetic variation of NOS-3, the gene encoding for eNOS, has been associated with a higher risk of development of cardiovascular diseases, such as hypertension and coronary heart disease (Sen et al., 2008; Matyar et al., 2005). Two of the most studied polymorphisms of NOS-3 are the 27-bp intron 4 repeat polymorphism (4VNTR) and the Glu298Asp missense mutation encoded by exon 7 of the NOS-3 gene. The 4VNTR polymorphism has been shown to segregate with lower plasma NO metabolites (Ignarro, 1990) and to be associated with RM in Caucasians (Tempfer et al., 2001). Moreover, a novel A>G single nucleotide polymorphism (SNP) in intron 6 has recently been reported to be associated with the risk of RM in a South Indian population (Suryanarayana et al., 2006). This SNP is very common in the Indian population, especially the South Indian population, and the proportion of the G (variant) allele in the homozygous condition was reported to be significantly different between the RM and the control groups.

Given that NO is necessary for the establishment and maintenance of pregnancy, this study investigated the role of eNOS in the aetiology of RM by investigating the distribution of eNOS 4VNTR, exon 7 Glu298Asp (g.12965G>T) missense mutation and intron 6 A>G polymorphism (g.12862A>G) in a group of 200 well-characterized North Indian women who had had at least three consecutive spontaneous first-trimester miscarriages of unknown aetiology. The association between polymorphism and RM was evaluated by comparing the genotyping results with a control group of 300 age-matched and ethnically related healthy parous controls with no history of complications of pregnancy or systemic diseases. Moreover, the distribution of three neighboring synonymous substitutions (g.12920C>T, g.12932C>T and g.13222C>T), previously known in other populations, are described for the first time in an Indian population. As far as is known, this is the first report from North India pertaining to eNOS gene polymorphisms and its association with RM.