Background
Preeclampsia, a pregnancy-specific syndrome affecting about 5% to 10% of all pregnancies, is characterized by hypertension and proteinuria after 20 weeks of gestation. It is a major cause of maternal and perinatal morbidity and mortality and occurs only in the presence of a placenta and remits dramatically after the placenta has been delivered. The underlying pathogenetic mechanisms of this maternal syndrome are much debated with the current hypotheses including inflammatory disease, vascular-mediated factors, placental ischemia, genetic predisposition and immune maladaptation [
1‐
3]. Preeclampsia is also associated with defective uteroplacental vascularization [
4] and impairment of angiogenesis and vascular transformation of the uteroplacental unit, which are crucial for normal fetal development [
5]. However, the molecular pathways responsible for normal angiogenesis and vascular remodeling in the fetomaternal unit are still poorly understood.
Vascular endothelial growth factor (VEGF) is a major angiogenic factor and plays an important role in all aspects of vascular development, including endothelial cell proliferation, migration, survival and regulation of vascular permeability [
6,
7]. VEGF and its two receptors, fms-like tyrosine kinase 1 (Flt-1) (VEGFR-1) and KDR/Flk-1 (VEGFR-2), have been shown to be part of an essential regulatory system for blood vessel formation. One of the two receptors, Flt-1 appears to have dual functions, with negative and positive activity in vascular endothelial cells. In early embryogenesis, Flt-1 functions as a negative regulator, most likely through its strong VEGF trapping activity [
8]. Flt-1 (-/-) mice are embryonic lethal due to the disorganization of blood vessels and overgrowth of endothelial-like cells within the lumens of blood vessels [
9]. In adult stages, however, Flt-1-specific ligand can induce a mild angiogenesis. Thus, this dual function may be tightly regulated and important for fine tuning the formation and maintenance of the blood vessel structure in placental vasculature.
During placental development, the expression of the Flt-1 gene is not only detected in vascular endothelial cells, but also in the developing trophoblasts [
10]. The Flt-1 gene is located in the chromosome region 13q12 and consists of 30 exons and 29 introns [
11,
12]. The gene encodes a high affinity receptor for VEGF, which has sixth immunoglobulin (Ig)-like domains in the extracellular region based on the distribution of cysteine residues, a transmembrane domain and an intracellular region containing a tyrosine kinase domain divided by a long kinase-insert domain [
11,
13]. There is a dinucleotide repeat in the 3' non-coding region of the Flt-1 gene [
11] that has been shown to be polymorphic [
14,
15]. This region of the gene codes for the intracellular part of the protein that is likely associated with signal transduction. Dinucleotide repeat regions are often used as disease markers and their functional significance is being increasingly realized [
16,
17]. Considering the important roles of Flt-1 in pregnancy, functional polymorphisms in the Flt-1 gene may be potentially important as genetic markers for susceptibility to preeclampsia. Based on genetic predisposition, this relationship may be strengthened by showing an association between polymorphisms of Flt-1 gene and an increased risk of developing preeclampsia. In view of the possible role of the Flt-1 gene in the etiology of preeclampsia, we investigated whether the dinucleotide (threonine-glycine; TG)
n repeat polymorphism in the 3' non-coding region of the Flt-1 gene is associated with preeclampsia in Korean pregnant women.
Results
Polymorphism analysis of the Flt-1 gene was performed for 170 preeclamptic patients and 202 normotensive pregnancies. The clinical characteristics of the study population are shown in Table
1. Maternal age, nulliparity, blood pressure, maternal weight at delivery, gestational age at delivery and birth weight were found to be significantly different between the two groups (
p < 0.05). In addition, 58 (34.12%) of the 170 preeclamptic patients delivered a fetus with IUGR, defined as birth weight below the 10th percentile for gestational age.
Table 1
Clinical characteristics of normal controls and preeclamptic patients
Maternal age (y) | 32.8 ± 3.7 | 30.9 ± 3.8 | < 0.001 |
Nulliparity (%) | 94 (46.5) | 139 (81.8) | < 0.001†
|
Systolic BP (mmHg) | 121.8 ± 10.2 | 159.2 ± 16.6 | < 0.001 |
Diastolic BP (mmHg) | 75.3 ± 8.7 | 100.5 ± 11.8 | < 0.001 |
Maternal weight at delivery (kg) | 68.0 ± 7.8 | 74.2 ± 10.3 | < 0.001 |
GA at delivery (wk) | 39.1 ± 1.3 | 36.5 ± 3.3 | < 0.001 |
Birth weight (g) | 3356.9 ± 436.1 | 2528.0 ± 776.4 | < 0.001 |
Proteinuria (dipstick) | - | 2.6 ± 1.0 | - |
IUGR (number) | - | 58 | - |
Ten alleles observed in this study groups were designated as allele*12 (A1) through allele*23 (A12), according to the number of TG repeats, which ranged in size from 102 bp (12 TG repeats with a 78 bp segment of amplified flanging sequences) to 124 bp (23 TG repeats) (Table
2). The frequency of the 14-repeat allele (A3) was most abundant (63.82% in preeclampsia and 69.06% in controls), followed by the 21-repeat allele (A10; 28.53% in preeclampsia and 23.76% in controls) (Table
2). The allele frequency was very similar to that previously reported for an American population [
14]. Interestingly, the 12-, 15-, 19-, and 20-repeat alleles had not previously been reported. However, the allele frequencies of the Flt-1 (TG)
n polymorphism in patients with preeclampsia did not differ from those in normal controls. Furthermore, the genotype frequencies in preeclampsia and controls did not significantly deviate from Hardy-Weinberg equilibrium (data not shown). The genotypes were classified into five groups; (TG)
14/(TG)
14, (TG)
14/(TG)
21, (TG)
21/(TG)
21, Other, Combination (Table
3). The most common genotype in normal controls (45.05%) was homozygous (TG)
14/(TG)
14, whereas those in preeclamptic patients (41.76%) was heterozygous (TG)
14/(TG)
21 (Table
3). We compared each reference group [(TG)
14/(TG)
14, (TG)
14/(TG)
21, or (TG)
21/(TG)
21 genotype] with Other (Table
3). Additionally, we also compared (TG)
14/(TG)
14 genotype with the combined genotype [(TG)
14/(TG)
21+(TG)
21/(TG)
21+Other] (Table
3). However, the genotype frequencies of the Flt-1 (TG)
n polymorphism in preeclamptic patients did not differ from those in normotensive pregnancies.
Table 2
Allele frequencies of the Flt-1 (TG)n polymorphism in normal controls and preeclamptic patients
A1 | 12 (102) | 2 (0.50) | 0 (0.00) |
A2 | 13 (104) | 4 (0.99) | 4 (1.18) |
A3 | 14 (106) | 279 (69.06) | 217 (63.82) |
A4 | 15 (108) | 3 (0.74) | 2 (0.59) |
A5 | 16 (110) | 1 (0.25) | 0 (0.00) |
A6 | 17 (112) | 0 (0.00) | 0 (0.00) |
A7 | 18 (114) | 0 (0.00) | 0 (0.00) |
A8 | 19 (116) | 5 (1.24) | 3 (0.88) |
A9 | 20 (118) | 2 (0.50) | 6 (1.76) |
A10 | 21 (120) | 96 (23.76) | 97 (28.53) |
A11 | 22 (122) | 8 (1.98) | 7 (2.06) |
A12 | 23 (124) | 4 (0.99) | 4 (1.18) |
Table 3
Genotype frequency of the Flt-1 (TG)n polymorphism in normal controls and preeclamptic patients
(TG)14/(TG)14
| 91 (45.05) | 65 (38.24) | 1.11 (0.59–2.09) | 0.746a
|
(TG)14/(TG)21
| 76 (37.62) | 71 (41.76) | 0.85 (0.45–1.60) | 0.613b
|
(TG)21/(TG)21
| 6 (2.97) | 11 (6.47) | 0.43 (0.14–1.35) | 0.143c
|
Other | 29 (14.36) | 23 (13.53) | - | - |
Combination | 111 (54.95) | 105 (61.77) | 1.32 (0.87–2.01) | 0.185d
|
Discussion
Microsatellites are powerful tools for performing linkage and association studies for diseases but they may also be directly involved in the modification of gene expression levels by silencing/enhancing transcription and modulating splicing events [
20,
21]. Since the microsatellite is located within intronic sequence with no obvious functional relevance, as shown by Turecki [
22], it is possible that specific alleles of this repeat may be in linkage disequilibrium with a nearby polymorphism that affects disease susceptibility. Moreover, variability in simple intronic repeats is probably involved in the etiology and pathogenesis of multifactorial diseases [
23].
However, although genetic factors have been extensively investigated during the past decade [
24], the dissection of the genetic basis for preeclampsia has been challenged by the wide clinical heterogeneity of this disorder and the lack of a full understanding of its underlying cause. The genes expressed in the placental vasculature throughout pregnancy are attractive candidate genes and nearly all accessible genes have been extensively analyzed. The Flt-1, a major receptor for VEGF, is produced as a 1338 amino acid residue precursor with a predicted 22 amino acid signal peptide. The extracellular domain of Flt-1 is composed of 736 amino acids; its transmembrane spanning domain is 22 amino acids and its intracellular domain is 558 amino acids. Spongiotrophoblast cells, endothelial cells and their progenitors are the major placental source of Flt-1 [
25,
26]. Given the regulatory role of Flt-1 in pregnancy and the presence of a multiallelic polymorphism in the Flt-1 gene, Flt-1 could conceivably be a candidate susceptibility gene in preeclampsia.
In this study, we focused on a d(TG)
n repeat polymorphism in the Flt-1 gene which may be associated with preeclampsia in Korean pregnant women. First, the Flt-1 gene contains a polymorphic TG dinucleotide repeat in the 3' non-coding region that might affect signal transduction. Secondly, decreased Flt-1 expression has been shown in the placental bed of preeclamptic patients [
27]. The down-regulation of Flt-1 in the placental bed may result in a decreased maternal vascular adaptation to pregnancy [
27]. Third, the production of the soluble, alternatively spliced Flt-1, sFlt-1, is significantly higher in preeclamptic patients compared with normotensive pregnant women [
28]. Finally, the embryos of Flt-1 mutant mice develop vasculature through endothelial cell differentiation. This vasculature is highly abnormal and unorganized with an overgrowth of endothelial cells crowding the lumen [
29].
Polymeropoulos [
14] first demonstrated that a higher percentage of 50 individuals from an American population had the 14-repeat allele of the (TG)
n polymorphism in the 3' non-coding region of the Flt-1 gene. They also observed that the distribution of the (TG)
n repeat alleles was bimodal, with two peaks at 14 repeats and 21 repeats, respectively. In this study, we tested, for the first time, the association between preeclampsia and the TG dinucleotide repeat polymorphism in the 3' non-coding region of the Flt-1 gene. Our data reveals that two alleles (14-repeat and 21-repeat) predominate in both preeclamptic and normal pregnancies, similar to the observation of Polymeropoulos [
14]. However, we could not find any differences in the allele or genotype frequencies of Flt-1 between preeclamptic patients and normal controls. This polymorphism of the Flt-1 gene was also studied by Parry et al. in minimal change nephropathy (MCN) patients compared to the standard American population [
19]. The investigators hypothesized that misregulation of Flt-1 may provide a mechanism for the development of protenuria in MCN, thus, polymorphisms in this gene may predispose to MNC. However, they did not demonstrate any deviation in the allele frequency in patients with MNC, implying that this locus does not contribute to susceptibility to MCN. Furthermore, we found that this dinucleotide polymorphism is not associated with a predisposition to preeclampsia. Alternatively, the penetrance of the Flt-1 gene may be modified by other factors, including distinct genetic loci that impart susceptibility.
Flt-1 can be activated by VEGF and placental growth factor (PlGF), which are highly expressed in the placenta. Most of the Flt-1 produced in the mouse and human placenta during later stages of gestation is the soluble form (sFlt-1) generated by alternative splicing of Flt-1, leading to a premature termination after the sixth Ig-like domain [
30]. sFlt-1 binds both VEGF and PlGF and acts as a soluble antagonist of their action. Flt-1 misregulation in peripheral blood mononuclear cells of pregnant women can result in the over-expression of sFlt-1, which may produce an additional (extra-placental) source of sFlt-1 that contributes to the etiology of preeclampsia [
31]. A recent report showed that levels of maternal sFlt-1 were elevated in preeclampsia and that administration of sFlt-1 to pregnant rats can cause symptoms of preeclampsia with glomerular endotheliosis [
32]. This idea clearly suggests that placental Flt-1 can play roles in regulating maternal vasculature during pregnancy. We have previously shown that sFlt-1 levels in the second trimester maternal plasma are significantly higher in women with preeclampsia than in normal pregnant women [
33]; however, we were unable to demonstrate an association between the d(TG)
npolymorphism in the 3' non-coding region of the Flt-1 gene and sFlt-1 levels.
Most common genetic disorders, such as preeclampsia, follow a complex mode of inheritance and may result from variants of many genes, each contributing only a weak effect to the disease. Common genetic polymorphism may explain a portion of the heritable risk for common diseases. Consequently considerable effort should be devoted to finding and typing common microsatellite polymorphisms in the human genome in order to understand the occurrence of relatively common phenotypes. As trophoblast cells, which also express Flt-1, are fetal of origin, the role of fetal Flt-1 (TG)
n polymorphism needs also to be examined in the risk of preeclampsia. Although we do not deny that one of the possible limitations of this case-control study is the relatively small sample size, we did take into account several issues that could lead to a false conclusion, such as established criteria for the diagnosis in order to exclude subphenotypes known to differ in the evaluation of the disease [
34] and matching of cases and controls for several risk factors and for genetic background.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
S–YK conceived the study, performed sequencing of the samples, and contributed to the analysis and interpretation of the data as well as to the writing of the manuscript. J–HL collected relevant clinical data and assisted in preparation of the DNA samples. J–HY, M–YK and JSC participated in collection of samples, clinical analyses, and clinical diagnosis. S–YP participated in collection of samples and revised the manuscript. H–MR developed the study design, was responsible for overall supervision of all aspects of this research project and revised the manuscript. All authors read and approved the final manuscript.