A Case-Control Study of Relevance Between NOS2 rs2297518 Polymorphism with Preeclampsia Risk in the Chinese Han women

Backgroud: Inducible Nitric Oxide Synthase (iNOS) acts on the contraction and expansion of blood vessels by mediating the synthesis of Nitric Oxide (NO), which is implicated in the pathophysiology of preeclampsia (PE) associated with systemic vasoconstriction. The polymorphism of NOS2, the gene of coding iNOS, can affect the function of protein. Therefore, we aimed to explore whether the polymorphism site rs2297518 in NOS2 is associated with susceptibility of PE in a Chinese Han population. Methods and Results:Genotyping the NOS2 rs2297518 polymorphism through TaqMan real-time uorescence quantitative polymerase chain reaction (PCR) after DNA extraction from blood samples in this case-control study including 979 PE patients and 1187 healthy pregnant women. Using independent sample t-test and chi-square test to analyse clinical data and experimental results. We performed the odds ratios (ORs) and 95% condence intervals (CIs) to estimate the degree of the association.There was no statistical signicance in the genetic distributions for the polymorphism of rs2297518 between the PE and control groups (c 2 =1.43, p=0.49 by genotype frequency, c 2 =0.85, p=0.36, odds ratio=1.07, 95% condence interval 0.92-1.25 by allele frequency), and no differences among early/late-onset or mild/severe PE and controls was seen. Conclusion: Our results indicated that the NOS2 rs2297518 polymorphism may be not play a major role in the susceptibility to PE in the Chinese Han population. Therefore, it is essential to test other polymorphisms of this gene to validate a potential relationship for susceptibility to PE. frequency PE patients population, the GA allele frequency and A allele frequency expresses an obvious increase in PE group. cohort obvious relationship between rs2297518 and the occurrence of hypertension[14]. the relevance between PE susceptibility and NOS2 rs2297518 from 979 PE patients and 1187 normal pregnant women in Chinese Han population, there were no signicant differences in genotypic and allelic frequencies of this SNP between PE and control groups. further understand the relationship between NOS2 and PE, divided the PE patients into mild/severe and early/late-onset groups, that there were also no signicant differences in genetic distributions at the polymorphic sites between mild/severe PE and control groups, or between early/late-onset PE and control groups. These data suggested that the rs2297518 in NOS2 may not play a pivotal role in the pathophysiology of PE in Chinese Han women. This study was the rst to explore the relevance between NOS2 rs2297518 and susceptibility of PE in the Chinese Han women. In conclusion, our results showed that the rs2297518 polymorphism in NOS2 was not associated with PE in Chinese Han women. Even though PE is a complex polygenetic hereditary disease and our research involves large-scale clinical data, the results may be inuenced by the multiple races and regions, which need to expand the sample size in a future study. In addition, we only explored the rs2297518 polymorphism in NOS2 but did not analyze the interaction with other polymorphisms. Therefore, genetic studies in other candidate genes for PE from different races and regions are required to detect the possibly different pathophysiological mechanisms, which will increase our understanding of the pathogenesis of PE and further to explore methods of targeted therapy relating to gene changes between populations.


Introduction
As a major cause of fetal growth restriction, preterm delivery and maternal death, preeclampsia (PE) is characterized by de novo development of hypertension and accompanied by proteinuria, which may progress into a multisystem disorder after 20 weeks gestation [1]. PE affects approximately 2~8% of all pregnant women in worldwide according to the World Health Organization. Although many therapeutic interventions have been proposed, the mortality and morbidity rates of PE patients remain as a challenge due to its unclear etiopathogenesis. The typical pathological manifestation of PE is systemic vasoconstriction, which may be related to abnormal changes in renal hemodynamics and NO level [2].
In normal physiological conditions, pregnant women have an obvious increase in renal plasma ow and glomerular ltration rate compared with ordinary women [3], which leads to the increased production of NO and expression of inducible NOS (iNOS) [4]. iNOS is a nitric oxide synthase, encoded by NOS2 and activated in in ammatory conditions by cytokines [5]. Besides, the expression of iNOS have been observed in villous stroma cells, which may play a protective role for the fetus to avoid infection [6]. However, other investigators have indicated that iNOS is involved in the occurrence of PE [7]. An animal test showed that increasing the iNOS expression of pregnant rats with placental ischemia led to vascular oxidative stress, and hypertension [7]. Besides, elevated vascular iNOS expression has been observed in the reduced uterine perfusion pressure(RUPP) rat model, a model that can present PE-like clinical symptoms [7]. When injected with an inhibitor of iNOS in this rat, PE features and increased level of iNOS was weaken [7]. Previous studies have indicated that the pathogenesis of placental ischemia in PE contributes to oxidative stress. In turn, reactive oxygen species (ROS) can react with NO produced by iNOS to form peroxynitrite in the development of PE [8,9]. Moreover, circulating pro-in ammatory cytokines (IL-1, IL-6, chemokine) can promote the increase of iNOS in PE patients compared to normal pregnancies[10]. This conclusion was also con rmed by another study that human umbilical vein endothelial cells (huvec) incubating with PE patient serum expressed higher iNOS mRNA compared with huvec stimulated by healthy pregnant women serum [11]. Analyzing the placenta iNOS expression of 32 PE patients and 32 healthy pregnant women, researchers found the iNOS placenta expression had an obvious increase in the PE case group [12]. However, Faxén et al. presented a different result that the iNOS placenta expression manifested a decrease trend in PE patients [13].
PE is a disease of multiple factors and aberrent genes leading to this pregnancy complication. In recent years, researchers have begun to focus on the associations between candidate genes with PE susceptibility. A functional polymorphic site of rs2297518, located in exon 6 of NOS2 [14], causes a base substitution from G to A at 2087 locos of its mRNA and leads to an amino acid change from serine to leucine (p.S696L), which can increase NOS2 activity by altering NOS2 protein function and confer higher NO production based on the A-allele [15]. Amaral [14]. Therefore, this study aimed to explore whether the rs2297518 polymorphism has statistical different between a PE group and a control group of Chinese Han women and further nd out the association between this polymorphism and susceptibility to PE. after 20 weeks of gestation, and accompanied by one of symptoms as follows: proteinuria (³0.3g/24h), urine protein/creatinine ratio ³0.3, random urine protein³(+) or when not accompanied by symptoms of proteinuria occurring upper abdominal discomfort, headache, and blurred vision, according to previously published criteria [17]. We excluded patients with a history of hypertension, diabetes mellitus, or systemic lupus erythematosus. Inclusion criteria for control were as follows: 1) no clinical history of PE, chronic hypertension, heart disease, kidney disease, diabetes mellitus, hepatic diseases, transfusion, or immunotherapy; 2) without obstetric complications such as premature membrane rupture, placenta previa, threatened abortion, arti cial insemination, twin or multiple pregnancies, and macrosomia in the present gestation. This study was approved by the Ethics Committee of the A liated Hospital of Qingdao University and obtained the written informed consent from all participants.

Materials And Methods
Taqman real-time uorescence quantitative PCR Total DNA was extracted from peripheral venous blood and NOS2 rs2297518 genetic distribution was genotyped by the TaqMan real-time uorescence quantitative polymerase chain reaction(PCR). The ampli cation method was performed according to previously published criteria [18]. The rs2297518 primers were 5'-GTTGAGCTCTTTCAGCATGAAGAGC -3' (forward) and 5'-ATTTCTTCAGTTTCTAGAAAGAGAG -3'(reverse). Through uorescence signal VIC and FAM to read results and then sanger sequencing to verify base represented by uorescent signal.
Statistical analysis SPSS 23.0 was used to data analysis. Student's t-test was used to compare differences in demographic and clinical characteristics between cases and controls. The chi-square test examined the Hardy-Weinberg equilibrium (HWE) in the control group to ensure that it was representative. Differences in genotypic and allelic frequencies between cases and controls were compared by Pearson's chi-square test.

Results
Demographic and Clinical Characteristics Table 1 showed a comparison of demographic and clinical characteristics between PE patients and control groups. There were no signi cant differences in maternal age and age of menarche between PE patients and control groups (p>0.05). However, compared to the controls, gestational age admission(weeks), gravidity(p=0.004), number of abortions(p=0.006), birth weight, blood pressure, diastolic blood pressure, white blood cell and neutrophils (p=0.003) of the PE group have signi cantly different (p<0.001).

Analysis of Genotypic and Allelic Frequencies
The control group in our study satis ed the HWE (rs2297518, c 2 =0.45, p=0.50). Table 2 showed the genetic distributions of NOS2 rs2297518 between PE and control groups. No signi cant differences were observed at this polymorphic site between the two groups in terms of genotypic distributions (χ 2 =1.43, p=0.49), nor for allelic frequencies (χ 2 =0.85, p=0.36, OR = 1.07, 95% CI 0.92-1.25).
To further investigate the relevance between NOS2 variant and PE, we divided PE group into severe PE group and non-severe PE group according to the diagnostic criteria of severe PE (pregnant women with PE have one of the following manifestations: systolic blood pressure ≥160 mmHg and/or diastolic blood pressure ≥110 mmHg; increased level of AST or ALT; urine protein quanti cation>2.0 g/24 h and so on) [17]. The samples with incomplete clinical data were not grouped and the number is 23. Early-onset PE patients were those diagnosed before the 34th week of gestation, and are known to be more severely affected than those with later-onset PE [19]. Similarly, there are 5 samples of incomplete clinical data were not grouped. Table 2 showed that there were also no signi cant differences in the genetic distributions of rs2297518 between early/late-onset PE and control groups (early-onset PE vs. control: χ 2 =1.90, p=0.39 by genotype, χ 2 =0.012, p=0.91 by allele; late-onset PE vs. control: χ 2 =1.82, p=0.40 by genotype, χ 2 =1.82, p=0.18 by allele).

Discussion
Preeclampsia is a serious pregnancy complication threatening the health of mother and fetus and can even cause death. Insu cient trophoblast cell invasion induces incomplete spiral artery remodeling and further leads to hysteroplacental hypoperfusion, which are main pathological manifestations of PE. The pathogenesis of PE may involve multiple pathological changes such as excessive systemic in ammation, oxidative stress, vasospasm, change of cytokines, but is currently unclear.
As an effective vasodilator, NO is involved in the blood pressure regulation of PE, and is activated by three NOSs including neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). iNOS and eNOS are found in placenta. Unlike the other two NOSs, iNOS can function independently of calcium or calmodulin [20]. The iNOS was found to express on villous mesenchymal cells by iNOS-speci c monoclonal antibody [21]. iNOS expression also can be detected in the vascular endothelial cells under the stimulation of in ammation [22]. Uncomplicated pregnancy is itself in the state of oxidative stress [23], active species superoxide will react with NO to produce peroxynitrite in the development of PE. iNOS can produce NO, thus involve the process of oxidative stress, which may be related to the occurrence of PE. Moreover, pregnancy itself will increase the expression of iNOS in smooth muscle cells, suggesting iNOS may be related to the regulation of uterine muscle tone [24]. iNOS expression is related to the release of free radicals and cytokines involved in PE [25,26]. Under the condition of in ammatory, iNOS may active more NO and be involve in the pathological process of PE. Besides, iNOS is concerned with the regulation of adaptive changes in renal hemodynamics during pregnancy, which may induce the increased blood pressure in PE [2]. Du [27]et.al suggested enhancive iNOS may facilitate endoplasmic reticulum stress that promoted apoptosis of placenta trophoblast cells in PE. Mazzant et.al [28] reported that the level of iNOS presented an evident increase in 30 women in a PE group compared with 30 healthy pregnant women. Besides, Mao et.al [29] found that apoErat and apoE-/iNOS-rat have an obvious increase in blood pressure compared with wild type (WT). What's more, the increase in blood pressure in the apoE-/iNOS-rat is higher than the apoE-rat, indicating iNOS may be involved in the pathogenesis of PE related to dyslipidemia.
The onset of PE is regulated by multiple genes, and the SNPs (single nucleotide polymorphisms)s of candidate genes were closely related to susceptibility of PE. Some SNPs of candidate genes related to oxidative stress such as XRCC1, CypA, SOD2 and others showed a statistically signi cant association with PE [30][31][32], among them, the polymorphisms of NOS2 play an important role in the development of PE [33]. BHATNAGAR et.al found G300A located in exon 8 and G274T in exon 16 of NOS2 were associated with the occurrence of PE [33] in the population of North India. However, NOS2 rs2779249(-1026C/A) could enhance transcriptional activity of the iNOS promoter and further promote the production of NO, although no obvious difference was found in this polymorphism between PE patients and healthy pregnancy women [34]. Therefore, we analyzed the rs2297518 polymorphism, located in the exon 6 of NOS2 in chromosome 17, that causes an amino acid substitution from serine to leucine and an increase in NOS2 activity, and even alters the NOS2 protein function [35]. Previous studies have shown this polymorphism could affect the susceptibility to many disease conditions, such as in ammatory bowel disease, migraine with aura and so on [35]. Bhatnagar et al. analyzed the polymorphisms of the NOS2 were associated with hypertensive disorders of pregnancy(HDP), especially with PE [35]. Amaral[8] analyzed the genotypes and alleles frequency of NOS2 rs2297518 in 212 healthy pregnant women and 187 PE patients of Brazil population, and found that the GA allele frequency and A allele frequency expresses an obvious increase in PE group. However, a Finland cohort study found no obvious relationship between rs2297518 and the occurrence of hypertension [14]. In our study, we explored the relevance between PE susceptibility and NOS2 rs2297518 from 979 PE patients and 1187 normal pregnant women in Chinese Han population, indicating that there were no signi cant differences in genotypic and allelic frequencies of this SNP between PE and control groups. To further understand the relationship between NOS2 and PE, we divided the PE patients into mild/severe and early/late-onset groups, the results showed that there were also no signi cant differences in genetic distributions at the polymorphic sites between mild/severe PE and control groups, or between early/late-onset PE and control groups. These data suggested that the rs2297518 in NOS2 may not play a pivotal role in the pathophysiology of PE in Chinese Han women. This study was the rst to explore the relevance between NOS2 rs2297518 and susceptibility of PE in the Chinese Han women.
In conclusion, our results showed that the rs2297518 polymorphism in NOS2 was not associated with PE in Chinese Han women. Even though PE is a complex polygenetic hereditary disease and our research involves large-scale clinical data, the results may be in uenced by the multiple races and regions, which need to expand the sample size in a future study. In addition, we only explored the rs2297518 polymorphism in NOS2 but did not analyze the interaction with other polymorphisms. Therefore, genetic studies in other candidate genes for PE from different races and regions are required to detect the possibly different pathophysiological mechanisms, which will increase our understanding of the pathogenesis of PE and further to explore methods of targeted therapy relating to gene changes between populations.