Polymorphisms of the endothelial nitric oxide synthase (NOS3) gene in preeclampsia: a candidate-gene association study
© Zdoukopoulos et al; licensee BioMed Central Ltd. 2011
Received: 12 August 2011
Accepted: 3 November 2011
Published: 3 November 2011
The endothelial nitric oxide synthase gene (NOS3) has been proposed as a candidate gene for preeclampsia. However, studies so far have produced conflicting results. This study examines the specific role of variants and haplotypes of the NOS3 gene in a population of Caucasian origin.
We examined the association of three common variants of the NOS3 gene (4b/a, T-786C and G894T) and their haplotypes in a case-control sample of 102 patients with preeclampsia and 176 women with a history of uncomplicated pregnancies. Genotyping for the NOS3 variants was performed and odds ratios and 95% confidence intervals were obtained to evaluate the association between NOS3 polymorphisms and preeclampsia.
The single locus analysis for the three variants using various genetic models and a model-free approach revealed no significant association in relation to clinical status. The analysis of haplotypes also showed lack of significant association.
Given the limitations of the candidate-gene approach in investigating complex traits, the evidence of our study does not support the major contributory role of these common NOS3 variants in preeclampsia. Future larger studies may help in elucidating the genetics of preeclampsia further.
Preeclampsia is a medical condition in which high blood pressure and elevated urinary excretion of protein develop in pregnancy . Family-based studies have shown that genetic factors may play a role in preeclampsia . In addition, candidate-gene association studies (GAS) on preeclampsia have not produced conclusive results so far . However, the pathogenesis of preeclampsia is poorly understood and the search for low-penetrance genes by hypothesis-driven candidate-gene studies (genetic association study-GAS) and hypothesis-free genome-wide association studies is ongoing .
The leading hypotheses, concerning the pathogenesis of preeclampsia, are based on disturbed placental function and impaired remodelling of the spiral arteries . Endothelial nitric oxide synthase (NOS3) is an important regulator of vascular tone and contributes to the reduction of the uteroplacental resistance seen in normal pregnancy [6–8]. Therefore, the endothelial nitric oxide synthase gene (NOS3), located at the 7q35-q36 region, has emerged as a logical candidate gene in the development of preeclampsia. Variants (polymorphisms) of the NOS3 gene have been investigated for association with preeclampsia and other disorders such as hypertension [9, 10]. The three most common variants examined for clinical relevance, based on their potential functional effects are : (i) a G894T substitution in exon 7 resulting in a Glu to Asp substitution at codon 298 (rs1799983), (ii) an insertion-deletion in intron 4 (4a/b) consisting of two alleles (the a*-deletion which has four tandem 27-bp repeats and the b*-insertion having five repeats), and (iii) a T786C substitution in the promoter region (rs2070744). A whole genome-scan meta-analysis for preeclampsia has already identified the locus of NOS3 gene as a promising candidate for preeclampsia susceptibility , although linkage studies seem to support a relationship between NOS3 and hypertension rather than preeclampsia [12, 13].
Previous GAS that investigated the association between NOS3 variants and preeclampsia have produced controversial or inconclusive results and the replication record of these studies is relatively poor [14, 15]. Therefore, the status of association for the NOS3 variants remains ambiguous. In this project we aimed to replicate previous findings on the association between the three most commonly investigated NOS3 polymorphisms (4b/a, T-786C and G894T) and preeclampsia, in a GAS conducted on a homogeneous population of Caucasian origin (Greeks). These variants were chosen because of their potential functional implications and their high minor allele frequency [16–18]. An analysis of haplotypes was also performed .
A total of 102 cases with preeclampsia and 176 female controls were recruited from the Department of Obstetrics and Gynecology, University Hospital of Larissa. Preeclampsia was defined as new hypertension (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg) and substantial proteinuria (>300 mg in 24 h) at or after 20 weeks' gestation . The study protocol was approved by the Institute Ethics Committee and all subjects provided a written informed consent. All controls faced pregnancies without complications and they were free of preeclampsia history. The controls were unrelated by blood to cases, and all apart from 3 (1.7%) were delivered at term. All study participants were of Caucasian origin whereas women with previous renal disease, diabetes, or history of metabolic disorders were excluded from the patient sample. A blood sample for biochemical measurements and DNA extraction was taken from each individual.
Genomic DNA was extracted from whole blood using the QIAamp DNA blood kit (QIAGEN, Valencia, CA, USA) following the manufacturer's instruction. Genotyping of each polymorphism was performed by amplification from 50 to 100 ng of genomic DNA. The primer sequences used and the laboratory conditions for genotyping (polymerase chain reaction, restriction enzymes, agarose electrophoresis) for each NOS3 polymorphism have been previously described [16–18]. Genotyping was performed by laboratory personnel blinded to clinical status.
Continues variables were compared using the Mann-Whitney non-parametric test. Categorical variables were compared using the Chi-square test. The genotypic distribution in the control group was tested for departing from the Hardy-Weinberg Equilibrium (HWE) and the existence of linkage disequilibrium (LD) between the two polymorphisms in both cases and control was tested by using exact tests according to Weir . The haplotype frequencies were estimated and compared using SHEsis . The association between genotype distribution and clinical status was tested using the chi-square test. The dominant and recessive models of the cases was compared to the control group using a logistic model. The significance of the genetic models was also tested using the Fisher's exact test. The comparisons associations were expressed in terms of odds ratio (OR), unadjusted and adjusted for age, with the corresponding 95% confidence interval (CI). The statistical analysis was performed using SPSS v11.5 (SPSS Inc.). A result was considered statistically significant when P < 0.05.
Characteristics of the study subjects.
30.64 ± 6.32
29.60 ± 5.18
35.41 ± 3.43
38.58 ± 2.14
Birth weight (g)
2266.5 ± 839
3078.0 ± 572
161.66 ± 10.3
115.84 ± 10.9
92.70 ± 8.47
70.40 ± 7.54
Distribution (%) of the NOS3 4b/a, T-786C and G894T genotypes among cases and control subjects.
(n = 102)
(n = 176)
ORG = 0.79 (0.48-1.29)
ORG = 0.93 (0.61-1.43)
ORG = 0.86 (0.56-1.32)
OR (95% CI)
ORadjusted (95% CI)
Allele contrast (b vs. a)
Allele contrast (C vs. T)
Allele contrast (T vs. G)
D', r2 and p-values for testing linkage disequilibrium between pairs of SNPs for cases and controls (in brackets).
D' = 0.66 (0.63)
r2 = 0.14 (0.15)
p = 0.02 (<0.01)
D' = 0.59 (0.54)
r2 = 0.04 (0.05)
p = 0.10 (0.11)
D' = 0.45 (0.38)
r2 = 0.17 (0.13)
p < 0.01 (<0.01)
Estimated haplotype frequencies for the three NOS3 polymorphisms (4b/a, T786C and G894T).
This study evaluated relations between common genetic variants and haplotypes in the NOS3 gene with preeclampsia. The single locus analysis among the three most commonly studied polymorphisms (4b/a, T-786C and G894T) revealed lack of association. The analysis of haplotypes also showed non-significant association.
The conclusion reached in the GAS was based on relatively small number of participants for each gene polymorphism and therefore, any inferences have to be cautious. Candidate-gene studies have a tendency to lack the power to detect weak genetic contributions of common variants [19, 23]. For example, in order to achieve a power >80% to identify a modest genetic effect (odds ratio 1.2) of a polymorphism present in 10% of individuals, a sample size of 10,000 subjects or more would be needed . Therefore, the sample sizes required to predict association have to be far beyond what is currently available and no single institution or entity alone will be able to provide a reasonable number of patients. Meta-analysis of multiple studies has clearly a role in offering an analysis with the potential for enhanced power .
It has been reported that the polymorphisms might be in linkage disequilibrium and interaction of the polymorphisms within haplotypes can be a major determinant of disease susceptibility than the individual polymorphism . Thus, individual NOS3 genotypes might not be reliable markers of risk for developing preeclampsia. The haplotype analysis approach is expected to be more powerful than single-marker analysis, because of the ancestral structure incarcerated in the distribution of haplotypes . Particularly if the markers used define mutations within functional DNA, then the haplotypes composed of these markers can have more of a biological role. In our analysis, we focused on haplotypes defined by the three most commonly investigated and potentially functional NOS3 variants (instead of other tested variants or HapMap tagging polymorphisms) because we aimed to replicate the already examined associations [9, 24, 25].
Interestingly, 13 studies [9, 24–35] have reported positive association for the variants NOS3 gene, whereas the two published meta-analyses and other individual studies did not replicate the findings [14, 15, 36, 37]. Thus, the available evidence from candidate-gene approach cannot support a major contributory role of NOS3 variants in preeclampsia pathogenesis. Since predicting the functional credentials of most genetic variants remains problematic, obtaining robust replication of positive association findings proves difficult . Moreover, the multifactorial etiology of common disorders involving complex epistatic and gene-environment interactions reduces the likelihood that a single type of studies, such as gene-candidate association studies, could provide conclusive inferences. An integration approach combining different kinds of genetic data analysis (i.e. genomic convergence of GAS, GWAS, genome-scans and microarrays) may be more effective in identifying valid genetic loci [39, 40].
In conclusion, the genetic association study did not replicate a significant association for the NOS3 variants and haplotypes. The results of the present individual GAS should be interpreted with caution since the number of participants were relatively small. However, preeclampsia is a complex disease with multifactorial etiology and therefore, a minor contributory pathogenetic role of NOS3 variants in conjunction with other genetic or environmental factors cannot be excluded . Collaborative research in preeclampsia may help in identifying the contributing role of variants by performing GAS and genome-wide association studies with adequate power . Furthermore, the design of rigorous studies for investigating epistatic and gene-environment interactions and the utilization of data generated by genomic studies may help in deriving more conclusive claims about the genetics of preeclampsia.
The single locus analysis and the analysis of haplotypes failed to demonstrate a significant association between preeclampsia and three common NOS3 polymorphisms.
This work was supported by the University of Thessaly in Greece. The authors wish to thank all the women who consented to be involved in the study.
- Lain KY, Roberts JM: Contemporary concepts of the pathogenesis and management of preeclampsia. JAMA. 2002, 287: 3183-3186. 10.1001/jama.287.24.3183.View ArticlePubMedGoogle Scholar
- Zintzaras E, Kitsios G, Harrison GA, Laivuori H, Kivinen K, Kere J, Messinis I, Stefanidis I, Ioannidis JP: Heterogeneity-based genome search meta-analysis for preeclampsia. Hum Genet. 2006, 120: 360-370. 10.1007/s00439-006-0214-1.View ArticlePubMedGoogle Scholar
- Ward K: Genetic factors in common obstetric disorders. Clin Obstet Gynecol. 2008, 51: 74-83. 10.1097/GRF.0b013e3181616545.View ArticlePubMedGoogle Scholar
- Roten LT, Johnson MP, Forsmo S, Fitzpatrick E, Dyer TD, Brennecke SP, Blangero J, Moses EK, Austgulen R: Association between the candidate susceptibility gene ACVR2A on chromosome 2q22 and pre-eclampsia in a large Norwegian population-based study (the HUNT study). Eur J Hum Genet. 2009, 17: 250-257. 10.1038/ejhg.2008.158.View ArticlePubMedGoogle Scholar
- Steegers EA, von Dadelszen P, Duvekot JJ, Pijnenborg R: Pre-eclampsia. Lancet. 2010, 376: 631-644. 10.1016/S0140-6736(10)60279-6.View ArticlePubMedGoogle Scholar
- Norris LA, Higgins JR, Darling MR, Walshe JJ, Bonnar J: Nitric oxide in the uteroplacental, fetoplacental, and peripheral circulations in preeclampsia. Obstet Gynecol. 1999, 93: 958-963. 10.1016/S0029-7844(99)00007-1.PubMedGoogle Scholar
- Sladek SM, Magness RR, Conrad KP: Nitric oxide and pregnancy. Am J Physiol. 1997, 272: R441-463.PubMedGoogle Scholar
- Magness RR, Shaw CE, Phernetton TM, Zheng J, Bird IM: Endothelial vasodilator production by uterine and systemic arteries. II. Pregnancy effects on NO synthase expression. Am J Physiol. 1997, 272: H1730-1740.PubMedGoogle Scholar
- Serrano NC, Casas JP, Diaz LA, Paez C, Mesa CM, Cifuentes R, Monterrosa A, Bautista A, Hawe E, Hingorani AD, et al: Endothelial NO synthase genotype and risk of preeclampsia: a multicenter case-control study. Hypertension. 2004, 44: 702-707. 10.1161/01.HYP.0000143483.66701.ec.View ArticlePubMedGoogle Scholar
- Zintzaras E, Kitsios G, Stefanidis I: Response to Endothelial Nitric Oxide Synthase Polymorphisms and Susceptibility to Hypertension: Genotype Versus Haplotype Analysis. Hypertension. 2006Google Scholar
- Zintzaras E, Kitsios G, Stefanidis I: Endothelial NO synthase gene polymorphisms and hypertension: a meta-analysis. Hypertension. 2006, 48: 700-710. 10.1161/01.HYP.0000238124.91161.02.View ArticlePubMedGoogle Scholar
- Arngrimsson R, Hayward C, Nadaud S, Baldursdottir A, Walker JJ, Liston WA, Bjarnadottir RI, Brock DJ, Geirsson RT, Connor JM, Soubrier F: Evidence for a familial pregnancy-induced hypertension locus in the eNOS-gene region. Am J Hum Genet. 1997, 61: 354-362. 10.1086/514843.View ArticlePubMedPubMed CentralGoogle Scholar
- Mitchell GF, DeStefano AL, Larson MG, Benjamin EJ, Chen MH, Vasan RS, Vita JA, Levy D: Heritability and a genome-wide linkage scan for arterial stiffness, wave reflection, and mean arterial pressure: the Framingham Heart Study. Circulation. 2005, 112: 194-199. 10.1161/CIRCULATIONAHA.104.530675.View ArticlePubMedGoogle Scholar
- Yu CK, Casas JP, Savvidou MD, Sahemey MK, Nicolaides KH, Hingorani AD: Endothelial nitric oxide synthase gene polymorphism (Glu298Asp) and development of pre-eclampsia: a case-control study and a meta-analysis. BMC Pregnancy Childbirth. 2006, 6: 7-10.1186/1471-2393-6-7.View ArticlePubMedPubMed CentralGoogle Scholar
- Medica I, Kastrin A, Peterlin B: Genetic polymorphisms in vasoactive genes and preeclampsia: a meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2007, 131: 115-126. 10.1016/j.ejogrb.2006.10.005.View ArticlePubMedGoogle Scholar
- Tesauro M, Thompson WC, Rogliani P, Qi L, Chaudhary PP, Moss J: Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: cleavage of proteins with aspartate vs. glutamate at position 298. Proc Natl Acad Sci USA. 2000, 97: 2832-2835. 10.1073/pnas.97.6.2832.View ArticlePubMedPubMed CentralGoogle Scholar
- Tsukada T, Yokoyama K, Arai T, Takemoto F, Hara S, Yamada A, Kawaguchi Y, Hosoya T, Igari J: Evidence of association of the ecNOS gene polymorphism with plasma NO metabolite levels in humans. Biochem Biophys Res Commun. 1998, 245: 190-193. 10.1006/bbrc.1998.8267.View ArticlePubMedGoogle Scholar
- Nakayama M, Yasue H, Yoshimura M, Shimasaki Y, Kugiyama K, Ogawa H, Motoyama T, Saito Y, Ogawa Y, Miyamoto Y, Nakao K: T-786-->C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation. 1999, 99: 2864-2870.View ArticlePubMedGoogle Scholar
- Zintzaras E, Lau J: Synthesis of genetic association studies for pertinent gene-disease associations requires appropriate methodological and statistical approaches. J Clin Epidemiol. 2008, 61: 634-645. 10.1016/j.jclinepi.2007.12.011.View ArticlePubMedGoogle Scholar
- Sibai B, Dekker G, Kupferminc M: Pre-eclampsia. Lancet. 2005, 365: 785-799.View ArticlePubMedGoogle Scholar
- Weir B: Genetic data analysis II: methods for discrete population genetic data. 1996, Sunderland MA: Sinauer AssociatesGoogle Scholar
- Shi YY, He L: SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci. Cell Res. 2005, 15: 97-98. 10.1038/sj.cr.7290272.View ArticlePubMedGoogle Scholar
- Zintzaras E, Lau J: Trends in meta-analysis of genetic association studies. J Hum Genet. 2008, 53: 1-9. 10.1007/s10038-007-0223-5.View ArticlePubMedGoogle Scholar
- Bashford MT, Hefler LA, Vertrees TW, Roa BB, Gregg AR: Angiotensinogen and endothelial nitric oxide synthase gene polymorphisms among Hispanic patients with preeclampsia. Am J Obstet Gynecol. 2001, 184: 1345-1350. 10.1067/mob.2001.115048. discussion 1350-1341View ArticlePubMedGoogle Scholar
- Chen LK, Huang CH, Yeh HM, Lee CN, Shyu MK, Hsieh FJ, Lai LP, Sun WZ: Polymorphisms in the endothelial nitric oxide synthase gene may be protective against preeclampsia in a Chinese population. Reprod Sci. 2007, 14: 175-181. 10.1177/1933719107299570.View ArticlePubMedGoogle Scholar
- Fatini C, Sticchi E, Gensini F, Genuardi M, Tondi F, Gensini GF, Riviello C, Parretti E, Mello G, Abbate R: Endothelial nitric oxide synthase gene influences the risk of pre-eclampsia, the recurrence of negative pregnancy events, and the maternal-fetal flow. J Hypertens. 2006, 24: 1823-1829. 10.1097/01.hjh.0000242407.58159.87.View ArticlePubMedGoogle Scholar
- Sandrim VC, Palei AC, Cavalli RC, Araujo FM, Ramos ES, Duarte G, Tanus-Santos JE: eNOS haplotypes associated with gestational hypertension or preeclampsia. Pharmacogenomics. 2008, 9: 1467-1473. 10.2217/146224126.96.36.1997.View ArticlePubMedGoogle Scholar
- Sandrim VC, Palei AC, Sertorio JT, Cavalli RC, Duarte G, Tanus-Santos JE: Effects of eNOS polymorphisms on nitric oxide formation in healthy pregnancy and in pre-eclampsia. Mol Hum Reprod. 2010, 16: 506-510. 10.1093/molehr/gaq030.View ArticlePubMedGoogle Scholar
- Seremak-Mrozikiewicz A, Drews K, Mrozikiewicz PM: The -786T/C polymorphism of the endothelial nitric oxide synthase gene in preeclampsia. Eur J Obstet Gynecol Reprod Biol. 2008, 138: 118-119. 10.1016/j.ejogrb.2007.02.008.View ArticlePubMedGoogle Scholar
- Seremak-Mrozikiewicz A, Drews K, Barlik M, Sieroszewski P, Grzeskowiak E, Mrozikiewicz P: The significance of -786T > C polymorphism of endothelial NO synthase (eNOS) gene in severe preeclampsia. J Matern Fetal Neonatal Med. 2011, 24: 432-436. 10.3109/14767058.2010.511329.View ArticlePubMedGoogle Scholar
- Sharma D, Singh A, Trivedi SS, Bhattacharjee J: Intergenotypic variation of nitric oxide and inflammatory markers in preeclampsia: A pilot study in a North Indian population. Hum Immunol. 2011Google Scholar
- Sharma D, Trivedi SS, Bhattacharjee J: Oxidative stress and eNOS (Glu298Asp) gene polymorphism in preeclampsia in Indian population. Mol Cell Biochem. 2011Google Scholar
- Tempfer CB, Dorman K, Deter RL, O'Brien WE, Gregg AR: An endothelial nitric oxide synthase gene polymorphism is associated with preeclampsia. Hypertens Pregnancy. 2001, 20: 107-118.View ArticlePubMedGoogle Scholar
- Yoshimura T, Yoshimura M, Tabata A, Shimasaki Y, Nakayama M, Miyamoto Y, Saito Y, Nakao K, Yasue H, Okamura H: Association of the missense Glu298Asp variant of the endothelial nitric oxide synthase gene with severe preeclampsia. J Soc Gynecol Investig. 2000, 7: 238-241. 10.1016/S1071-5576(00)00060-5.View ArticlePubMedGoogle Scholar
- Aggarwal PK, Jain V, Jha V: Endothelial nitric oxide synthase, angiotensin-converting enzyme and angiotensinogen gene polymorphisms in hypertensive disorders of pregnancy. Hypertens Res. 2010, 33: 473-477. 10.1038/hr.2010.23.View ArticlePubMedGoogle Scholar
- Hillermann R, Carelse K, Gebhardt GS: The Glu298Asp variant of the endothelial nitric oxide synthase gene is associated with an increased risk for abruptio placentae in pre-eclampsia. J Hum Genet. 2005, 50: 415-419. 10.1007/s10038-005-0270-8.View ArticlePubMedGoogle Scholar
- Tempfer CB, Jirecek S, Riener EK, Zeisler H, Denschlag D, Hefler L, Husslein PW: Polymorphisms of thrombophilic and vasoactive genes and severe preeclampsia: a pilot study. J Soc Gynecol Investig. 2004, 11: 227-231. 10.1016/j.jsgi.2003.12.002.View ArticlePubMedGoogle Scholar
- Hattersley AT, McCarthy MI: What makes a good genetic association study?. Lancet. 2005, 366: 1315-1323. 10.1016/S0140-6736(05)67531-9.View ArticlePubMedGoogle Scholar
- Kitsios GD, Zintzaras E: Genome-wide association studies: hypothesis-"free" or "engaged"?. Transl Res. 2009, 154: 161-164. 10.1016/j.trsl.2009.07.001.View ArticlePubMedPubMed CentralGoogle Scholar
- Kitsios GD, Zintzaras E: Genomic convergence of genome-wide investigations for complex traits. Ann Hum Genet. 2009, 73: 514-519. 10.1111/j.1469-1809.2009.00533.x.View ArticlePubMedPubMed CentralGoogle Scholar
- Clayton D, McKeigue PM: Epidemiological methods for studying genes and environmental factors in complex diseases. Lancet. 2001, 358: 1356-1360. 10.1016/S0140-6736(01)06418-2.View ArticlePubMedGoogle Scholar
- Pearson TA, Manolio TA: How to interpret a genome-wide association study. JAMA. 2008, 299: 1335-1344. 10.1001/jama.299.11.1335.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/11/89/prepub
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