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Susceptibility to preeclampsia is associated with a 50-bp insertion/deletion polymorphism at the promoter region of the SOD1 gene

Objective: There is much evidence that oxidative stress is involved in the pathogenesis of preeclampsia (PE). A 50 bp insertion/deletion (Ins/Del) functional polymorphism in the promoter region of SOD1 has been reported. Due to a total lack of data, the aim of this study was to investigate the association between the SOD1 (Ins/Del) polymorphism and the risk of PE. Material and Methods: The current hospital-based case-control study included a total of 172 preeclamptic and 171 non-preeclamptic pregnancies. Genotyping was performed using the polymerase chain reaction method. Results: Statistical analysis revealed that the Del/Del genotype significantly correlated with susceptibility to PE [odds ratio (OR): 6.53, 95% confidence interval (CI): 1.43-29.7, p=0.015]. Since maternal body mass index, family history of PE in first degree relatives, and educational levels were statistically associated with the susceptibility to PE, further analyses were carried out in order to estimate the adjusted ORs. After adjustment for aforementioned variables, the Del/Del genotype increased the risk of PE (OR: 5.98, 95% CI: 1.21-29.5, p=0.028). Conclusion: The 50 bp Ins/Del in promoter region of the SOD1 gene could be an intriguing susceptibility factor for developing preeclampsia in Iranian Caucasians.


Introduction
Preeclampsia (PE) affects approximately 5-8% of pregnancies and greatly contributes to morbidity/mortality of mother and fetus (1). Preeclampsia is defined as proteinuria and hypertension after 20 weeks of gestation. Furthermore, preeclamptic women are at risk for developing cardiovascular disease (2). The detailed etiology of PE remains unknown and thus precise prediction and prevention are difficult. Genetic components and environmental factors are known to be involved in the etiology of PE, and therefore PE is highly heritable (3,4). Many genetic association studies of candidate genetic polymorphisms have been performed to investigate the genetic background of PE (5).
Reactive oxygen species (ROS) lead to oxidation of numerous biomolecules, including DNA (6). Oxidative stress can occur due to an imbalance between production of ROS and antioxidant capacity. There is plentiful evidence that such conditions are implicated in the pathogenesis of PE (4,7-9). A 50 bp insertion/deletion (Ins/Del) in the SOD1 promoter region has been reported (10). This is a functional polymorphism and alters gene expression with the Del allele resulting in lower SOD1 mRNA levels (11). Association between the SOD1 expression level and PE has been investigated (12). SOD1 expression is significantly down-regulated under oxidative stress, for example it is decreased in morphine treated human SH-SY5Y cells (13), and in cells exposed to electromagnetic fields (14). SOD1 Ins/Del genetic variation is related with complex human diseases associated with oxidative stress, such as breast and gastric cancers, age of onset of bipolar disorder, and cardiovascular disease (15)(16)(17)(18)(19).
We hypothesized that the Ins/Del genetic variation in SOD1, which is involved in cellular detoxification, may represent a good candidate for susceptibility to PE. Since no data has been published on the association between the SOD1 (Ins/ Del) polymorphism and the risk of PE, a hospital-based, casecontrol study was carried out and is presented here.

Participants
This was a hospital-based case-control study. A total of 172 preeclamptic and 171 non-preeclamptic pregnancies, as healthy control group, were included in the study. The participants were recruited from the delivery ward at Zainabeieh, Hafez and Dena Hospitals (Shiraz, Fars province, south-west Iran). DNA extraction, genotype determination and statistical analysis were done in our research laboratory. The preeclamptic and non-preeclamptic women were matched with each other by age. PE was defined as persistent blood pressure above 140/90 mmHg and proteinuria of more than 0.3 g/24 hours, developing after 20 weeks of gestation. Exclusion criteria were twin pregnancies, fetal growth retardation, altered renal function, recurrent miscarriages, and placental abruption. The patients were not classified according to the severity of PE.
Body mass index (BMI, kg/m 2 ) was defined as weight (kg) divided by square of height (m 2 ). Data on age, family history of PE among first-degree relatives, educational level, gravidity, physical activity, interval from last pregnancy, and smoking habits were obtained through interviews with participants. As the gene pool of the Iranian population is heterogeneous (20,21), participants were selected from Caucasian Persian/ Muslims living in Fars province, south-west Iran.
The minimum sample size that would be necessary to identify a significant difference in genotype distribution between PE and normal pregnancies was estimated using QUANTO software (http://biostats.usc.edu/foftware). Assuming 15% frequency for the D allele, equal number of cases and controls, Rg: 1.8, a=0.05, and b=0.80, minimally, 149 patients would be required. In the present study 172 PE and 171 normal pregnancies were included.
The current study was approved by the Ethical Committee of Shiraz University (approval number: SU.DB-9630578). This study was conducted in accordance with the Declaration of Helsinki. All pregnant women signed an informed consent before the study.

Genotyping
DNA was extracted from whole blood using a previously described method and stored at -20 °C until use (22). Genotyping analysis was carried out based on a polymerase chain reaction (PCR) method using specific primers as described previously (19). The PCR reaction was performed using 0.4 pmol/mL of each primer, 1.5 mM of MgCl 2 , 0.2 mM each of desoxynucleotide triphosphates, 0.12 U/mL of Taq DNA polymerase (SinaGene, Iran) and genomic DNA as template. The reaction was preincubated for 5 minutes at 95 °C for denaturation, followed by 35 seconds at 95 °C, 30 seconds at 60 °C, and 35 seconds at 72 °C, for 31 cycles and a final extension at 72 °C for 10 minutes. The amplified DNA was electrophoresed in 1.5% agarose gel, under constant current of 90 volts for 40-60 minutes. Gels were stained and visualized by ethidium bromide staining under ultraviolet illumination.

Statistical analysis
Hardy-Weinberg equilibrium was investigated in the healthy controls and PE patients by c 2 analysis. Continuous clinical characteristics were compared between the PE cases and non-PE controls, using Student's t-test. We performed unconditional logistic regression analysis to estimate odds ratios (ORs) and 95% confidence intervals (95% CI) for assessment of differences in the prevalence of characteristics between PE cases and non-PE controls.

Results
Descriptive characteristics of the PE and control groups are shown in Table 1, 2, containing continuous and binary variables, respectively. Preeclamptic mothers compared to control subjects had significantly higher BMI (p<0.001) ( Table  1). The mean interval from last pregnancy was statistically similar between normal pregnancies and PE outcomes. A positive history for PE among first degree relatives (OR: 8.25, 95% CI: 3.39-20.0, p<0.001) and higher educational levels (OR: 3.77, 95% CI: 2.01-7.08, p<0.001) were associated with risk of PE. However, neither smoking nor gravidity were associated with PE (Table 2). A higher percentage of controls had some history of smoking compared to the PE group, but due to the very low frequency of smokers, the difference was not significant. Figure 1 shows the genotypes of the study polymorphism by electrophoresis of the PCR products on 1.5% agarose gel. The Ins/ Del alleles have 297 and 247 bp, respectively. Table 3 summarizes the distribution of genotypes of the SOD1 Ins/Del genetic variation in PE and normal pregnancies. The genotypes were in Hardy-Weinberg equilibrium in control subjects (c 2 =0.02, df=1, p=0.880). However, there was significant deviation between observed and expected genotypic frequencies in the PE group (c 2 =11.9, df=1, p<0.001). Statistical analysis indicated that the Del/Del genotype was significantly correlated with susceptibility to PE (OR: 6.53, 95% CI: 1.43-29.7, p=0.015). The Del allele increased the risk of PE (OR: 1.67, 95% CI: 1.08-2.58, p=0.020).
The risk of PE significantly increased as a function of the number of Del alleles (c 2 for trend: 4.75, p=0.029).
Since maternal BMI, family history of PE in first degree relatives, and educational levels were statistically associated with the risk of PE, further analyses were carried out in order to estimate the adjusted ORs. After adjustment for these variables, the Del/Del genotype significantly increased the susceptibility to PE (OR: 5.98, 95% CI: 1.21-29.5, p=0.028).

Discussion
These results showed that maternal BMI, interval from last pregnancy, family history of PE among first degree relatives, and educational levels were significantly associated with PE, which were similar to previous studies (1,4,5,(23)(24)(25).
It is well established that oxidative stress is associated with the pathogenesis of PE (4,7-9). SOD1 is involved in the antioxidant system and it has several genetic polymorphisms, including the 50 bp Ins/Del functional polymorphism in its promoter region (10). Investigation of the association between the SOD1 Ins/ Del functional polymorphism and the risk of PE was the main aim of this study. The Del allele is reported to significantly decrease promoter activity (11). Therefore, it was suggested that individuals having the Del/Del genotype would have lower antioxidant capacity compared to the Ins/Ins genotype (11). The Del/Del genotype showed positive correlation with susceptibility to PE, which supported our hypothesis. Previously, reduced expression of SOD1 in peripheral blood mononuclear cells of PE patients had been reported (12). In addition, a significantly lower level of the SOD1 mRNA in trophoblast cells isolated from placentas of PE pregnancies has been reported (26,27