Introduction

Preeclampsia (PE) is a common multisystem and multifactorial pregnancy disorder that affects approximately 2%–8% of all pregnancies. Despite the developments in prenatal and neonatal care, this disorder is still one of the most important reasons behind maternal and neonatal morbidity and mortality [1]. PE is characterized by systolic blood pressure (SBP) ≥ 140 mm Hg or diastolic blood pressure (DBP) ≥ 90 mm Hg with evidence of proteinuria ( ≥ 300 mg/24 h) after 20 weeks of pregnancy. Other symptoms include edema, vision defects, headache, abdominal pain, and thrombocytopenia [2, 3]. Moreover, women suffering from PE have an increased risk of cardiovascular disorders that suggests a common etiology [4]. Although the etiology and pathogenesis of PE have not been clearly identified, it is thought to have an essential genetic component [5, 6] Several genetic case–control studies have stated associations between PE and various gene polymorphisms [7,8,9,10]. The lack of reproducibility of these studies has led to the uncertainty about the genes that contribute to the disease pathogenesis [11]. Several studies have demonstrated that inappropriate regulation of the renin–angiotensin system (RAS) may contribute to the pathogenesis of multiple cardiovascular disorders and blood pressure regulation during pregnancy [12, 13]. Angiotensin-converting enzyme (ACE), a key component of the RAS, converts inactive angiotensin I into active angiotensin II [14]. The ACE gene (Gene ID: 1636) is located in chromosome 17q23 and comprises several polymorphisms including Insertion/Deletion (I/D) and rs4343 that alter the enzyme activity [15, 16]. The I/D polymorphism is characterized by a 287 base pair segment that is either inserted (I) or deleted (D) in the ACE gene intron 16. The consequence of rs4343 polymorphism, located in exon 17, is Thr 776 Thr synonymous substitution (NP_000780.1).

As studies on the etiology of PE have suggested an inherited susceptibility, any relation between PE and genetic polymorphisms of the components of the RAS-related genes will be of interest.

Therefore, in the present study, we assessed the association of the ACE gene I/D and rs4343 variations with PE in Iranian women. We believe that this is the first study regarding the association between rs4343 variation and PE susceptibility in Iranian population.

Methods

Subjects

In this case–control study, 296 participants were enrolled, including 165 PE patients and 131 healthy controls with similar age and ethnic background. The mean age was 33.69 ± 8.92 years (range: 17–42) and 34.5 ± 10.03 years (range: 16–40) in the patients and controls, respectively. The criteria defined for PE were as SBP ≥ 140 mm Hg or DBP ≥ 90 mm Hg measured at least two times after 20 weeks of pregnancy, and the existence of proteinuria ≥ 300 mg/24 h or ≥ 1 + reading on dipstick in a random urine sample at least twice with no evidence of a urinary tract infection.

Severe PE patients had SBP ≥ 160 mm Hg, DBP ≥ 110 mm Hg, and a proteinuria level of > 5 g/24 h or > 3 + . Patients who had gestational hypertension, history of hypertension, diabetes mellitus, and autoimmune and endocrine diseases were not included in the present study. The normotensive control subjects were recruited from the same center randomly and had no history of pregnancy complications. The same exclusion criteria were considered for the control group. We obtained informed written consent from all participants. The study protocol was approved by Shahid Beheshti University of Medical Sciences ethics committee (Code No: IR.SBMU. MSP.REC.1396.792).

Genotyping of I/D polymorphism

After collecting whole blood samples from all subjects in EDTA tubes, genomic DNA was extracted by using the BioFact Genomic DNA Prep Kit (Catalog number GD261-060, South Korea) according to the manufacturer’s procedure.

I/D polymorphism was detected with conventional PCR analysis according to the previous study [17]. The 25 μl amplification reaction included 12.5 μl Taq DNA Polymerase 2 × Master Mix Red (Ampliqon, Denmark), 5 pmol/l of each primer, and genomic DNA ( ≥ 100 ng). PCR was performed on a GeneTouch thermal cycler apparatus (BIOER, China) and the amplification program was as follows: initial denaturation for 1 min at 94 °C; 30 cycles at 94 °C for 40 s, 67 °C for 30 s, and 72 °C for 40 s; and final extension for 4 min at 72 °C. The amplified PCR products were electrophoresed on a two percent agarose gel prepared in 0.5 × Tris-Borate-EDTA buffer. The PCR amplicons were 192 and 480 bp for the alleles with deletion and insertion, respectively. The accuracy of genotyping was further confirmed with Sanger sequencing of 10% of the DNA samples using an Applied Biosystems 3730xl DNA Analyzer (Macrogen, Korea).

Genotyping of rs4343 polymorphism

To genotype rs4343, tetra primer-amplification refractory mutation system-PCR (TP-ARMS-PCR) method was carried out according to a previous study [18]. The components of the 25 µl PCR reaction solution were as follows: 12.5 μl of Taq DNA Polymerase 2 × Master Mix Red (Ampliqon), 3 pmol/l of forward outer, 5 pmol/l of reverse outer, 12 pmol/l forward inner and 10 pmol/l of reverse inner primers, and ≥ 100 ng of genomic DNA. GeneTouch thermal cycler apparatus (BIOER) was used to amplify genomic DNA and the amplification program was as follows: initial denaturation for 1 min at 94 °C; 30 cycles at 94 °C for 35 s, 61 °C for 45 s, 72 °C for 35 s, and a final extension for 4 min at 72 °C. The amplified PCR products were electrophoresed on a two percent agarose gel prepared in 0.5 × Tris-Borate-EDTA buffer. The rs4343 A and G alleles created 202 and 134 bp bands, respectively. The outer primers amplified a common 280 bp band. The accuracy of genotyping was further confirmed with Sanger sequencing of ten percent of the samples by using an ABI 3730xl DNA analyzer (Macrogen).

Statistical analysis

We used SNPStats online software for calculating allele and genotype frequencies for both variations (http://bioinfo.iconcologia.net/SNPstats) [19]. Deviation from the Hardy–Weinberg equilibrium (HWE) was estimated by using the χ2-test. The association with PE was investigated in recessive and dominant models. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated to define the strength of association between the studied polymorphisms and susceptibility to PE. The P-value of < 0.05 was considered to be statistically significant.

Results

Table 1 describes the characteristics and demographic data of the study contributors. The SBP and DBP values were significantly higher in PE patients compared with controls (P < 0.05). No significant difference was observed in pre-pregnancy body mass index (BMI) between the studied groups (P > 0.05). Moreover, the patients had a significantly lower gestational age at delivery and the fetal birth weight compared with the controls (both P < 0.05). A higher proportion of the patients had a history of pregnancy loss (OR = 2.75, 95% CI, 1.49–5.06; P = 0.001) compared with normal subjects. The same situation was observed in the family history of hypertension between the studied groups (OR = 2.29, 95% CI, 1.36–3.83; P = 0.002). In total, 63.64% of the patients were diagnosed as mild PE, whereas 36.36% were severe PE. The distribution of all genotypes was in complete HWE in the control group for both studied polymorphisms. Table 2 displays the genotype and allele frequencies for I/D and rs4343 polymorphisms in the studied groups.

Table 1 Clinical and demographic features and relationship between risk factors and PE in the cases and controls
Full size table
Table 2 The I/D and rs4343 polymorphisms genotype and allele frequencies in the cases and controls
Full size table

For the rs4343, the G allele frequency was significantly higher in the case group (59.1%) compared with the control group (43.1%) (OR = 1.90, 95% CI, 1.37–2.65; P = 0.0001). The frequency of AG (OR = 2.36, 95% CI, 1.28–4.37; P = 0.006) and GG (OR = 2.89, 95% CI, 1.92–4.36; P < 0.0001) genotypes were different between PE patients and healthy subjects. Moreover, the genotype frequencies were different in the studied groups under dominant (OR = 3.94, 95% CI, 2.05–7.56; P < 0.0001) and recessive (OR = 2.21, 95% CI, 1.22–4.01; P = 0.009) inheritance models. For the I/D polymorphism, we did not observe significant differences in the genotype and allele frequencies or any of the inheritance models between the PE patients and controls.

Discussion

PE is a multifactorial disorder with a strong genetic evidence in the development of the disease. As a major component of the RAS, ACA has a key role in salt and water homeostasis and blood pressure [20]. Polymorphisms in the ACE gene affect the enzyme serum and tissue levels and therefore may play a significant role in PE. An increased level of ACE activity is considered a key factor in blood pressure alteration because of the increase in the potent vasoconstrictor angiotensin II and inactivation of bradykinin as a vasodilator factor [21]. The present results showed that the rs4343 is associated with PE in our studied population. Previous studies have revealed that rs4343 polymorphism is associated with left ventricular hypertrophy, blood pressure, coronary artery diseases and migraine [18, 22, 23]. However, there are only two studies regarding the role of rs4343 in developing PE [24, 25]. Zhang et al. demonstrated that fetal but not maternal rs4343 was associated with PE in Han Chinese women [24]. Procopciuc et al. [25] also revealed that the rs4343 increased the risk of PE; however, this increase was not statistically significant.

The rs4343 results in the Thr 776 Thr synonymous substitution in the ACE gene (NP_000780.1). Emerging evidence suggests that silent mutations could have functional consequences and therefore contribute to the human disease risk [26]. On the mRNA level, these types of mutations can affect stability, folding and translation rate. In addition, they can result in aberrant mRNA splicing. It is obvious that any alterations in the mRNA molecule will affect the related protein structure, expression, substrate specificity, secretion and/or enzymatic activity [26, 27]. The main reason that a silent mutation may change protein properties is the codon usage bias, which refers to the preferred use of particular codons instead of other synonymous codons during mRNA translation. The frequency of codon usage varies among species, tissues, and genes [28].

To determine the effect of rs4343 on mRNA folding and the kinetics of local translation, we performed two in silico prediction analysis. First, the effect of the rs4343 on local stem-loop mRNA structure of ACE gene was predicted by the RNAsnp Web Server online program [29]. Second, we measured the change in relative synonymous codon usage (RSCU) value that explains possible alterations in the kinetics of local translation as previously described [26]. Although the mRNA folding was not predicted to be significantly affected by the rs4343 variation; however, the vale of RSCU decreased from 1.14 to 0.46 (ΔRSCU = − 0.68) (For further details, please see the Supplementary File S1). This reduction might be associated with a slower local rate of translation elongation on the rare codon compared to the wild-type and therefore could affect the enzyme properties. Moreover, it is suggested that rs4343 alters regulatory motifs for several transcription factors and therefore affects the ACE levels [18]. Zhu et al. [30] showed that the rs4343 had the greatest significant effect on the ACE function and the presence of the G allele increased both SBP and DBP. It seems that the rs4343 G allele increases activity of serum ACE and therefore produces higher levels of angiotensin II. Moreover, this allele was also associated with higher ACE serum levels in a sample of depressed patients [16].

There was no association between ACE I/D alleles or genotypes and PE in our studied population, and this is consistent with several but not all previous reports. There are controversies considering the results obtained from the previous studies regarding the role of ACE I/D in PE [20, 21, 31,32,33,34]. It is suggested that the DD genotype is associated with higher levels of ACE enzyme, blood pressure, and increased risk of cardiovascular disorders [35,36,37]. González-Garrido et al. [21] reported that ACE II and DD genotypes are protective and risk factors for PE in Mexican women, respectively. They concluded that the D allele increases the disease risk [21]. Although the frequency of the DD genotype in the case group was higher than in controls in the present study, however the difference was not statistically significant (Table 2). It seems that the existence of a silencer-like sequence in the I allele results in a greater ACE level in the D allele [38]. Zhang et al. [24] also confirmed the association of ACE I/D polymorphism with PE in Han Chinese population; however, the same as rs4343, this association was found in fetal but not maternal samples [24]. In a meta-analysis study, Serrano et al. suggested that the detected increase in the PE risk associated with the D allele is due to small-study bias [39]. Totally, the observed inconsistency among previous studies is due to genetic heterogeneity, ethnicity, sample volume, PE criteria, and other environmental factors.

Investigation of the role of ACE gene polymorphisms in PE maybe a reliable tool to identify and manage the women at risk. Alvi et al. [40] showed that the combination of the AA and DD genotypes results in higher than average blood pressure level, both in hypertensive and healthy people in the Punjabi population.

In conclusion, the present results suggest the association between rs4343 and susceptibility to PE. Further educations with a larger sample size and analyses of genetic and environmental factors are needed to confirm the present results. In addition, measuring the enzyme serum levels may prove the functional effects of the studied polymorphisms.

Summary

What is known about this topic?

  • Deregulation of the renin–angiotensin system play an important role in the pathogenesis of preeclampsia.

  • Polymorphisms in the ACE gene affect the enzyme serum and tissue levels.

  • Previous studies indicated controversies considering the association between ACE I/D gene polymorphism and susceptibility to preeclampsia.

What this study adds?

  • To the best of our knowledge, this is the first study that reports maternal ACE rs4343is associated with preeclampsia.

  • The rs4343 alters regulatory motifs for several transcription factors and therefore affects the ACE level.

  • No association was detected between ACE I/D alleles or genotypes and PE in the studied population.