Objective
To determine whether polymorphisms in the FOXP3 gene are associated with preeclampsia.
Study Design
Case-control study in which 120 women with preeclampsia were compared with 120 healthy normotensive controls. Genetic variants (single nucleotide polymorphisms and microsatellites) in the FOXP3 gene were analyzed. Polymorphisms were chosen based on studies of the FOXP3 gene in other autoimmune disorders. Correction of P values for multiple comparisons was performed by using the Benjamini-Hochberg procedure.
Results
There were no differences in the genotypes or allele frequencies in the single nucleotide polymorphisms between cases and controls. The FOXP3 GT microsatellite allele at 266 bp was less common in cases than controls (1.0% vs 5.2%, P = .0264). However, this did not remain significant after correction for multiple comparisons.
Conclusion
Preeclampsia is not associated with FOXP3 gene polymorphisms that have been associated with other autoimmune disorders.
Preeclampsia remains a major cause of both maternal and perinatal morbidity and mortality complicating approximately 5% of pregnancies; 2-3% have clinically severe disease. It is one of the most common complications of pregnancy and is seen disproportionately in women with their first viable pregnancy. The pathophysiology of preeclampsia may include an underlying autoimmune or coagulopathic process, aberrations in the renin-angiotensin system, or failure of adequate angiogenesis, including endothelial dysfunction. However, despite a significant research effort on multiple fronts, we still have not elucidated the mechanism by which preeclampsia develops.
Normal pregnancy requires a relative maternal immune tolerance of the fetus. We know that women are at increased risk of preeclampsia in their first pregnancy, and with new partners. Decreased exposure to semen from the father of the child, preconception also has resulted in an increased risk of preeclampsia. Taken together, these observations have led to the hypothesis that preeclampsia may in part be immune mediated. This article focuses on the possibility of an immune component in the development of preeclampsia, and specifically, the role of regulatory T-cells (Tregs) and the Forkhead Box P3 (FOXP3) gene.
Tregs are a subset of T-cells that are thought to be immunosuppressive. Decreased numbers of circulating Tregs have been reported in women with pregnancy complications, including recurrent pregnancy loss and preeclampsia. Tregs are thought to be produced by upregulation of the FOXP3 gene that results in the conversion of naïve T-cells to Tregs. The FOXP3 gene is located at Xp11.23. It has been demonstrated in mouse models that inactivation of FOXP3 results in a lack of Tregs and notable organ-specific autoimmunity. Less is known about the function of FOXP3 in humans.
Other investigators have reported an association between FOXP3 gene polymorphisms and a variety of autoimmune disorders, including autoimmune thyroid disease, type I diabetes, and systemic lupus erythematosus. We therefore undertook a study to determine whether polymorphisms in the FOXP3 gene are associated with preeclampsia.
Materials and Methods
This was a case-control study comparing 120 women with preeclampsia and 120 healthy normotensive controls. All women who had previously consented to participate in a universal biobanking protocol were considered potential study participants. They were included as cases if they met standard clinical criteria for preeclampsia, including systolic blood pressure ≥140 or diastolic blood pressure ≥90 with evidence of proteinuria (1+ on urinalysis or ≥300 mg on a 24-hour urine collection). All controls were noted to be normotensive throughout their pregnancy, intrapartum, and immediately postpartum. Women with a history of autoimmune disorder, including thyroid disease, diabetes, systemic lupus erythematosus, or rheumatoid arthritis, were excluded. Women with known underlying renal disease were also excluded.
All women gave written informed consent for participation in a separate universal biobanking protocol. University of Utah Institutional Review Board approval was then obtained for the use of these samples in our study.
Demographic data were abstracted from the neonatal and maternal medical records. Specific maternal variables included age, race, parity, presence of preeclampsia, hypertension (gestational or chronic), gestational age at delivery, and delivery route. Maternal medical histories were also reviewed to ensure that the study subjects did not have any other known autoimmune disorders.
DNA was prospectively collected as part of a universal banking protocol for later retrospective comparisons. The blood (5 mL) was collected in EDTA tubes. Samples were centrifuged and the plasma and buffy coat were removed and stored at −80°C. DNA was extracted from samples by using established methods (Puregene; Qiagen Systems, Valencia, CA).
Genetic variants in the FOXP3 gene that had previously been assessed in other autoimmune disorders were analyzed. Three microsatellites [(GT)n; (TC)n; DXS573] associated with autoimmune thyroid disorders, a single nucleotide polymorphism (SNP) that has been associated with SLE (rs12843496), as well as 6 additional SNPs previously tested in patients with juvenile idiopathic arthritis (rs6609857, rs2294020, rs2280883, rs2232367, rs3761547, and rs4824747) were assessed in cases and controls. These specific polymorphisms were chosen in an effort to screen the entire FOXP3 gene. The microsatellite associated with type I diabetes in the Bassuny et al study was associated with decreased luciferase expression. However, the other chosen polymorphisms have not been investigated for a specific functional role.
Individuals were genotyped for the SNPs tested using TaqMan allelic discrimination chemistry (Applied Biosystems, Foster City, CA) with validated primers according to the manufacturer’s protocols. Specifically, 6 ng of DNA was included in a 5 μL reaction with a final concentration of 1X Taqman genotyping master mix and 0.5X Taqman genotyping assay mix. The genotyping assay mix was specific to each SNP and contained both primers and allele specific probes. After a 10-minute initial denaturation at 95°C, samples were amplified for 40 cycles of 95°C for 15 seconds and 60°C for 1 minute on an ABI 9700 thermal cycler (Applied Biosystems). Genotypes were detected through endpoint analysis. Genotyping software (SDS V2.3; Applied Biosystems) was used to automatically determine sample genotypes and generate cluster plots. Genotypes were subsequently manually verified, and SNPs were evaluated for deviation from Hardy-Weinberg Equilibrium.
For the microsatellites, 20 ng of DNA was amplified in a final volume of 50 μL using the polymerase chain reaction (PCR) and published primers. The forward primer of each microsatellite was labeled on the 5′ end with the fluorescent molecule 5/6 FAMM. Amplification was under the following conditions: 1X TaqGold master mix (ABI), 4 mm MgCl 2 , 2.5 mM/dNTP, 0.5 μM/primer, and 1.25 units of Amplitaq Gold DNA polymerase. Amplification involved initial denaturation at 94°C for 10 minutes, followed by 40 cycles of 94°C 45 seconds, 55°C 30 seconds, 72°C for 90 seconds, followed by a final extension at 72°C for 10 minutes. The amplified product was subjected to fragment analysis using capillary electrophoresis and an internal lane standard, to normalize allele calls, on an Applied Biosystems 3730XL. Fragment sizes and genotypes were determined with GeneMapper software V 4.0 (Applied Biosystems) that automatically assigns fragment sizes to the observed peaks.
The χ 2 contingency tables were used to compare the allele and genotype frequencies between cases and controls for the SNPs and the allele frequencies in the microsatellites. Microsatellite allelic association was analyzed as a dichotomous trait such that the observed frequency of the alleles between cases and controls were compared with the observed frequency in cases and controls of all other alleles.
The SNPs and microsatellites were all chosen based on previous published studies in which these specific gene variants in the FOXP3 gene were investigated for a role in other autoimmune disorders. The 3 microsatellites were assessed using 28 different alleles. Given that we performed a genetic study, where a relatively large number of microsatellite alleles were examined, we used the Benjamini-Hochberg procedure to adjust the P values. This maintains the false discovery rate (FDR) to no larger than the nominal alpha .05 level. In such studies, controlling for multiplicity in the standard fashion, such as with the Bonferroni procedure that controls the family-wise error rate (FWER) is not justified, whereas control for the FDR provides the correct control for multiplicity.
Student t test was used to compare demographic characteristics between groups for continuous variables. The χ 2 was used for dichotomous variables. Statistical analyses were performed in STATA 11.0 (Stata Corp, College Station, TX), and statistical significance was defined as a 2-sided P value < .05.
Results
One hundred twenty women with an antepartum diagnosis of preeclampsia who had specimens available for DNA analysis were included. Fifty-two (41.6%) women had severe preeclampsia, 68 (54.4%) had mild preeclampsia, and 5 (4.0%) had HELLP syndrome. The 120 controls were normotensive, parous women. Controls had no reported history of autoimmune disease. Five of the cases had chronic hypertension and were diagnosed with superimposed preeclampsia. As expected, cases were more likely than controls to deliver preterm, be nulliparous, and require a cesarean delivery ( Table 1 ).
