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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Author Contributions
  9. References

Pre-eclampsia (PE) is a multifactorial pregnancy-specific vascular disorder characterized by hypertension and proteinuria and affects around 3–8% of pregnancies worldwide. Defective placentation during the early stage of pregnancy most likely in combination with maternal and environmental factors could lead to systemic inflammation, endothelial dysfunction and the manifestation of the clinical symptoms. Inadequate number of regulatory T cells (Tregs) or their functional deficiency is linked with infertility, miscarriage and PE. It is well identified that forkhead box P3 (Foxp3) gene is a master control gene for the development and function of Tregs that play an important role in the maintenance of self-tolerance and mediate maternal tolerance to the foetus. The main objective of this study was to assess the maternal susceptibility to PE with respect to a deletion mutation in exon-2 and -3279 C > A polymorphism (rs3761548) in the promoter region within the Foxp3 gene in a total of 282 PE patients and 215 normal pregnant women. The results showed that exon-2 deletion mutation is present in 1.06% of patients and none in the controls, indicating that it was not a common gene polymorphism associated with PE. With respect to rs3761548, the C allele frequency was observed to be higher in patients than in controls (49% versus 27%; OR = 2.81, P < 0.01). In conclusion, our results are suggestive of A allele to be protective against PE and C allele as predisposing in a dose-dependent manner in our population.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Author Contributions
  9. References

Pre-eclampsia (PE) is a pregnancy-specific disorder recognized from antiquity as a leading cause of maternal and perinatal mortality that affects 3–8% [1] of pregnancies. It can manifest as a maternal syndrome with hypertension, proteinuria and oedema or a foetal syndrome with placental growth restriction, reduced amniotic fluid and abnormal oxygenation. Together, pre-eclampsia is characterized by inability of the trophoblasts to invade the decidual arteries leading to alterations in placental development, placental perfusion and an insufficient transport of nutrients to foetus [2]. The aetio-pathogenesis of PE is not yet fully understood. It has been suggested that deficient implantation may lead to infertility, whereas inappropriate placentation to foetal growth restriction. However, pre-eclampsia results from placental dysfunction that alters maternal system progressing through a preclinical to a clinical stage ultimately leading to maternal manifestations of the disease [3]. Abundant evidence suggests that the involvement of immunological factors is crucial in the causation of PE.

One of the main problems observed early in pre-eclampsia is an altered regulation of the immune system, where the shift towards Th2 cytokine profile observed in normal pregnancies does not occur. Various components of the immune system may be participating in the aberrant immunological alterations in the early placental microenvironment, impaired angiogenesis, as well as changes in local oxygen tension activation that pathologically affect early pregnancy events [4]. Regulatory T (Treg) cells play a crucial role in the maintenance of self-tolerance, physiological immune responses and also mediate maternal tolerance to the foetus. Elevated levels of Tregs in maternal blood and deciduas are associated with normal pregnancy, whereas decreased number of circulating Tregs has been reported in women with pregnancy complications, including pregnancy loss and pre-eclampsia. Maturation, development and function of Treg cells depend on the expression of transcription factor Foxp3 and cytokines and regulation of other T cell subsets through the NF-AT/NF-kB and Smad7 pathways. The Foxp3 gene is a member of the forkhead/winged helix family of transcriptional regulators whose dysfunction results in hyperactivation of T cells. It is located on the X-chromosome (Xq11.23–Xq13.3), consists of 11 exons and encodes a protein (431aa) with a molecular weight 47.25 kD. It contains four functional domains, a proline-rich repressor domain at N terminal end followed by a Zinc finger, Leucine zipper and a conserved Forkhead domain.

Role of Foxp3 was first reported by Bennett CL et al. [5] in IPEX (Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome), an autoimmune disease. Zhou Jianjun et al. [6] published that Th1 type immunity was predominant in pre-eclampsia with decreased expression of Foxp3 mRNA. The genetic variants of Foxp3 gene have not been explored sufficiently in terms of maternal susceptibility to pre-eclampsia. This study focuses on deletion mutation in exon-2 [7], which leads to frameshift mutation that is associated with abolition of functional domains of Foxp3, and a promoter SNP (rs3761548) that is related with downregulation of Foxp3 transcript [8] to see for the maternal susceptibility to pre-eclampsia in South Indian women. To the best our knowledge, this is the first report on pre-eclampsia with respect to deletion mutation and SNP rs3761548 of Foxp3.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Author Contributions
  9. References

Study population.  Present investigation was conducted on a total of 497 individuals recruited from Gandhi and J.J. Hospitals, Hyderabad, India, which included 282 PE patients and 215 ethnically matched healthy pregnant women. All the controls were normotensive throughout their pregnancy. Demographic data including age, parity, age at first conception, gestational age, type of delivery and liver function tests were recorded from the patient’s medical records. Patients were selected for the study based on their clinical symptoms and the diagnosis made by gynaecologists. All the subjects were made to understand the reasons for the sample collection, and written consent was obtained before taking the samples. Ethical approval was acquired from institutional ethics committee (Osmania University).

Inclusion criteria.  Patients were recruited based on International Society For The Study Of Hypertension In Pregnancy (ISSHP) that defines PE as hypertension with systolic blood pressure ≥140 mm Hg and diastolic blood pressure ≥90 mm Hg after 20 weeks of gestation together with proteinuria ≥300 mg in a 24-h collection or, ≥1+ on dipstick testing not associated with urinary tract infection or ruptured membranes.

Exclusion criteria.  Patients who had chronic hypertension, diabetes, polycystic ovary syndrome or the index pregnancy with a multifoetal gestation or conceived by assisted reproductive technology or with premature preterm rupture of membranes or with unexplained vaginal bleeding or the usage of antihypertensive drugs were excluded from this study.

DNA extraction and genotyping.  Five millilitre of peripheral blood samples was collected in EDTA vacutainers from all the subjects and stored at −20 °C until use. Genomic DNA was isolated by salting out method using standard protocol. DNA purity and concentration were determined by spectrophotometric (eppendorff) measurements of absorbance at 260 and 280 nm. All the samples were genotyped for the Foxp3 promoter −3279 A > C polymorphism and exon-2 deletion mutation where the reactions were carried out in a final volume of 20 μl reaction mixture containing: 2.5 μl of 100 ng DNA, 2.5 μl MgCl2, 1.25 μl of Taq DNA polymerase (Labpro, Hyderabad, India), 1 μl of each primer, 2.5 μl dNTPs (Labpro) and 1 μl PCR buffer.

Foxp3 Exon-2 deletion mutation.  Primers were designed to amplify the exon-2 region encompassing the deletion mutation (Del A) at 76th codon. The primers used for this mutation were as follows F: 5′-TAGCATCTACCATGTGGGCTT-3′ and R: 5′-TGAGGTGTTACCAGGTGGGA-3′. In brief, the PCR conditions were an initial denaturation at 94 °C for 4 min, followed by 35 cycles at 94 °C for 30 s, annealing at 56 °C for 30 s and extension at 72 °C for 30 s. The final extension was at 72 °C for 7 min and hold was at 4 °C. The amplified products (451 bp) were digested with BfaI restriction endonuclease to yield the fragments of 116 and 335 bps for wild-type allele and undigested 451 bp fragment for mutant allele on separation by electrophoresis with 2% agarose gel, stained with ethidium bromide (Fig. 1).

image

Figure 1.  Gel electrophoresis image of the Foxp3 promoter region (−3279 C > A) through allele-specific PCR.

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Foxp3 -3279 C > A polymorphism.  For this SNP, genotypes were determined by using ARMS PCR with sequence-specific primers (PCR-SSP), A-allele-specific primers, forward 5′-CTGGCTCTCTCCCCAACTGA-3′; reverse:5′-CAGAGCCCATCATCAGACTCTCTA-3′ and C-allele-specific primers forward :5′-TGGCTCTCTCCCCAACTGC-3′; reverse 5′-ACAGAGCCCATCATCAGACTCTCTA-3′. The PCR-amplified product sizes were 333 bp and 334 bp for A and C alleles respectively. The procedure for PCR include an initial denaturation at 94 °C for 4 min, denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 30 s, then step 2–4 for 30–40 cycles, extended extension at 72 °C for 7 min and hold at 8 °C. The amplified products were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide and photographed (Fig. 2).

image

Figure 2.  Gel electrophoresis image of Foxp3 Exon-2 deletion mutation.

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Statistical analysis.  The chi-square contingency tables were used to compare the allelic and genotypic frequencies between cases and controls. The risk associated with individual alleles or genotypes was calculated as the odds ratio with 95% confidence interval by using online 2 × 2 contingency calculator. In addition, Hardy–Weinberg equilibrium (HWE) test was evaluated by chi-square for the allelic frequency in both patients and controls for its deviation. Statistical analysis was carried out by using spss version 14 (spss.en.softonic.com) wherever required. Statistical significance was defined as a two-sided P-value <0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Author Contributions
  9. References

Foxp3 deletion mutation in exon-2

None of the 215 normal subjects included in this study showed this deletion mutation. Among the patient group, 279 of 282 demonstrated the normal allele and 3 of 282 (1.06%) exhibited the deletion in heterozygous condition. However, there were no homozygotes observed for deletion. RFLP and sequencing of the PCR product have confirmed the deletion in the patients. This deletion leads to a frameshift and predicts a premature termination codon at position 109, resulting in a truncated protein product of 108 amino acids compared with 431 residues in the wild type.

Foxp3 promoter −3279 C > A polymorphism

Table 1 shows the frequency of each genotype in the PE and healthy women. The frequencies of AA, AC and CC genotypes among patients and controls were 27.66%, 47.52%, 24.82% and 54.42%, 36.74%, 8.84%, respectively. A statistically significant difference was observed between the frequencies of Foxp3 genotypes in the two groups. With respect to homozygotes, CC genotype was predominant in PE patients and AA in controls (P < 0.05). However, the frequency of heterozygotes (AC) did not differ between the two groups. In addition to this outcome, when the three individual genotypes were compared with other two genotypes independently, the following results were obtained: (1) CC versus other genotypes OR = 3.4062 (CI 1.9791–5.8623, P < 0.01); (2) AA versus others OR = 0.32 (CI 0.2203–0.4676 P > 0.05); (3) AC versus others OR = 1.56 (1.8846–2.24, P < 0.01). In addition to these results, when each genotype is compared with other genotype, highly significant P-values were obtained (P < 0.01) with regard to their odds ratios (Table 2).

Table 1. Distribution of genotype and allele frequencies of FOXP3 promoter (−3279 C > A) polymorphism in controls and pre-eclampsia patients.
CategoryGenotypic frequencies N (%)Allelic frequency
CCACAACA
CONTROLS (215)19 (8.84)79 (36.74)117 (54.42)0.270.73
PATIENTS (282)70 (24.82)134 (47.52)78 (27.66)0.490.51
Table 2. Odds ratio of FOXP3 promoter region (−3279 C > A) genotypes in controls and pre-eclampsia patients.
CategoryOdds ratio (95% CI)
  1. *P < 0.05; **P < 0.01; NS, Non-significant.

AA versus Others0.3203 (0.2203–0.4657)NS
AC versus Others1.5587 (1.0846–2.24)*
CC versus Others3.4062 (1.9791–5.8622)*
AA versus AC0.393 (0.264–0.585)**
AC versus CC0.4604 (0.260–0.817)**
AC versus AA2.5443(1.708–3.790)**
CC versus AA5.526 (3.099–9.847)**
CC versus AC2.172(1.223–3.852)**
AA versus CC0.181 (0.1011–0.324)**

The allelic frequencies of A and C were 0.51 and 0.49 in patients and 0.73 and 0.27 in controls. The risk of PE in women with C allele was observed to be higher than the controls (49% versus 27%). In both patients and controls, the allelic frequencies were in accordance to Hardy–Weinberg equilibrium (P > 0.5).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Author Contributions
  9. References

Pre-eclampsia has been termed as the ‘disease of theories’ because of the confusion surrounding its aetiology. Hypertension and proteinuria that are relied on to define pre-eclampsia may simply be the final common pathway through which more than one type of pathology is being expressed. Among all the other changes that a woman’s body undergoes during pregnancy, her immune system also goes through adaptations, as the maternal immune system plays a part in the placentation process as well as in the maintenance of pregnancy. It was suggested that regulatory T cells support pregnancy maintenance by suppressing placental inflammation, while diminished T regulatory function may accompany reproductive failure [9]. Foxp3, a transcription factor, expressed by T regulatory cells was realized to be a candidate gene for autoimmune diseases because of its role in immunoregulation that was based on the involvement of Foxp3 mutations/alterations in IPEX [10]. Development of Treg cells requires continuous expression of Foxp3, while altered expression may result in its functional deficiency [11, 12]. Several functional polymorphisms of Foxp3 gene have been explored in the pathogenesis of various human diseases [13, 14]. In the present study, a deletion mutation in exon-2 and a functional polymorphic marker rs3761548(C > A) of Foxp3 have been investigated in PE patients and normal pregnant women of south India.

In this study, deletion mutation in exon-2 was observed in three of 282 (1.06%) PE patients in heterozygous condition, but none of them were homozygotes. However, this deletion was not detected in any of the control subjects suggesting that it is not a common polymorphism in the population. In a Japanese study dealing with IPEX, Foxp3 mutation was identified in the proband, with a single base deletion at codon 76 of exon-2, leading to a frameshift, which predicted a truncated protein product. The significance of the Foxp3 frameshift mutation would be predicted to lead to the absence of the essential forkhead DNA-binding domain and, hence, a null allele.

In addition to the deletion mutation analysis, Foxp3 C > A (rs3761548) polymorphism has been investigated in this study. Previous studies have reported that A allele of this polymorphism was observed to be a risk factor for autoimmune disorders such as systemic lupus erythmetosus [15], autoimmune thyroid diseases [16] and allergic rhinitis [17]. Shen Z et al. [8] have demonstrated that A allele causes loss of binding to the e47 and c-myb factors, leading to defective transcription of Foxp3 gene in patients with psoriasis, and AA genotype has been shown to be associated with lowest production of Foxp3 among the other genotypes. However, increased risk for psoriasis was reported in individuals with AC (−3279 C > A) genotypes with respect to this promoter polymorphism compared with CC genotype in Han Chinese population [13]. No association was shown in a study by Ogyi Park et al. [14] on chronic inflammatory bowel disease with respect to this SNP.

With regard to reproductive disorders in relation to Treg cells, many studies have shown that these cells are essential for maternal tolerance of the conceptus [18]. Treg cells accumulate in the deciduas and are elevated in maternal blood from early in the first trimester. Inadequate expression of Foxp3 is associated with recurrent spontaneous abortions (URSA), unexplained infertility and recurrent implantation failure. A study from Chinese Han population showed significant association of URSA patients with rs3761548, suggesting patients with AA genotype may have fewer Treg cells and/or weaker suppressive function thereby difficult to achieve foetal tolerance leading to early foetal loss [19]. However, no association was found with endometriosis-related or idiopathic infertility [20]. A recent study by Metz et al. [21] examined the role of Foxp3 gene polymorphisms in several SNPs other than rs3761548 among PE patients and controls and found no association. Comparative studies on PE with regard to this SNP are not available in the literature.

In our study, women with CC genotype exhibited 3-fold risk (OR = 3.40; P < 0.05) and individuals with AC genotype showed 1.5-fold (OR = 1.56; P < 0.05) possibility of developing PE, indicating the dose-dependent effect of C allele. This dose-dependent protective and predisposing effects of A and C alleles were highly prominent when odds ratio was calculated between homozygotes (Table 2). Therefore, based on the present results, C allele is suggested to be a predisposing allele that may contribute to the pathogenesis of PE in our population. AA genotype, a condition suggestive of pro-inflammatory, is predominant in normal pregnant women (54.41%), thus appears to be protective, whereas the frequency of this genotype is reduced to 27.65% in the patients (OR = 0.32; P > 0.05). Jenny et al. [22] suggested that moderate Th1 activity is a part of early normal pregnancy, consistent with a mild inflammatory environment controlled by Treg cells.

The maternal immune system is involved throughout the maintenance of pregnancy beginning from placentation and stays active and modified throughout the whole pregnancy. Optimally, it maintains protective responses towards harmful pathogens, but selectively decreases harmful Th1 and pro-inflammatory reactions, and increases the beneficiary Th2 and anti-inflammatory responses directed towards the foetus. On the other hand, genetic variations may be responsible for inadequate shift in Th1/Th2 that may result into PE, a multifactorial condition that is because of both immunological and angiogenic factors.

Based on our results, we hypothesize that because of C > A SNP in Foxp3 gene, there may be an anti-inflammatory response right from the initial stage of the pregnancy, which results in large number of Treg cells, an event that is expected to happen in the mid of the pregnancy. However, to confirm this hypothesis, diverse SNPs in relation to Tregs at various stages of pregnancy should be quantified. Further, to understand the significance of C allele association with PE found in this study, the role of Foxp3 gene polymorphisms and its interaction with other Treg cell–associated markers such as IL-10, IFN-γ, CTLA-4, etc., should also be taken into consideration in future studies. To our knowledge, this is the first study to investigate the association of Foxp3 SNP and the exon-2 deletion mutation in pre-eclampsia.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Author Contributions
  9. References

We extend our thanks to UGC for financial support [F.No.34-217/2008 (SR)]. We are grateful to all the patients and controls of JJ hospital, Gandhi Hospital, Hyderabad, for their cooperation in providing blood samples and clinical information.

Author Contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Author Contributions
  9. References

Parveen Jahan developed the concept and prepared the manuscript, Roopa Sreenivasagari performed experimentation, Deepthi Goudi carried out sampling, Prasanna Latha Komaravalli applied statistical analysis and Ishaq.M carefully read the manuscript and incorporated his valuable suggestions.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Author Contributions
  9. References
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