Mol. Hum. Reprod. Advance Access originally published online on February 4, 2005
Molecular Human Reproduction 2005 11(3):207-210; doi:10.1093/molehr/gah151
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An A>G polymorphism at position –670 in the Fas (TNFRSF6) gene in pregnant women with pre-eclampsia and intrauterine growth restriction
1First Department of Obstetrics and Gynecology, Semmelweis University Medical School, Budapest, Hungary and 2Division of Immunology and Infectious Diseases, Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, New York, NY, USA
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, 525 East 68th Street, Box 35, New York, NY 10021, USA. Email: switkin{at}med.cornell.edu
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Abstract |
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Fas-mediated apoptosis of maternal lymphocytes during pregnancy has been postulated to prevent the development of pre-eclampsia. A single adenine (A) to guanine (G) polymorphism at position –670 in the Fas gene (TNFRSF6) results in decreased Fas synthesis. The association between this polymorphism and pre-eclampsia in Hungarian women was investigated. In a case–control study, buccal swabs from 38 pregnant women with pre-eclampsia and 89 normotensive controls were analysed for the TNFRSF6–670 polymorphism. Investigators were blinded to clinical outcomes. Maternal homozygosity for the TNFRSF6–670*A occurred in 33 (37.1%) normotensive pregnant women as compared to only 5 (16.1%) of 31 pre-eclamptic pregnant women who delivered at <37 weeks gestation (P=0.04). The carriage rate of the TNFRSF6–670*G variant was also higher among these patients (59.7%) than among normotensive controls (42.1%; P=0.01). There was no relation between the polymorphism and the pre-eclampsia diagnosed at
37 weeks. Among pre-eclamptic patients with an intrauterine growth restriction (IUGR) neonate, eight (57.2%) were TNFRSF6–670*G homozygous as opposed to 3 (17.6%) of 17 pre-eclamptics who did not have IUGR (P=0.03) and 19 (21.3%) normotensive controls (P=0.008). Carriage of the TNFRSF6–670 polymorphism in the neonate was not associated with pre-eclampsia or IUGR. Maternal possession of the TNFRSF6–670*G increases the risk for pre-eclampsia and pre-eclampsia-associated IUGR in women who deliver at <37 weeks. Key words: Fas/genetic polymorphism/intrauterine growth restriction/pre-eclampsia/TNFRSF6
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Introduction |
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Hypertensive disorder is the most common medical complication during pregnancy affecting approximately 5–8% of pregnant women (Walker, 2000
). In USA, pregnancy-associated hypertension is directly responsible for 17.6% of maternal deaths (Koonin et al., 1997). Moreover, pregnancy-induced hypertension is a major cause of fetal morbidity and mortality (American College of Obstetricians and Gynecologists, 2002).
Recent studies suggest that pre-eclampsia may be more than one disease (Vatten and Skjaerven, 2004). In one third of cases, pre-eclampsia is accompanied by restricted fetal growth, probably originating from disturbed implantation of the conceptus. Placental abnormalities might lead to impaired uteroplacental blood flow resulting in placental hypoxia, reduced fetal nutrition and intrauterine growth restriction (IUGR). On the other hand, delivery of an appropriate or high birthweight neonate may indicate a spectrum of hypertensive conditions, ranging from mild pre-eclampsia with modest placental involvement to gestational hypertension without placental disease. These latter forms of hypertension are mainly diagnosed only at or close to term.
Despite efforts at early diagnosis, defining risk factors, aetiologies and pathogenic mechanisms as well as optimizing the management of pre-eclampsia remain unclear. Recent observations support the role of an immune-mediated pathogenesis in this disorder involving a disturbance in programmed cell death (apoptosis) in the placenta (Allaire et al., 2000). A major mediator of apoptosis is the Fas ligand—Fas (CD 95) system. Fas is a transmembrane receptor and a member of the tumor necrosis factor (TNF) receptor family. When Fas ligand binds to Fas, a series of intracellular events is triggered culminating in the irreversible activation of the apoptotic cascade (Griffith et al., 1995). Fas is not expressed on the surface of resting T lymphocytes, but is prominent on activated T cells (Zhang et al., 1997). Fas ligand is found at many immunologically privileged sites and is also expressed on human trophoblast cells throughout gestation (Bamberger et al., 1997). It has been hypothesized that the trophoblast-associated Fas ligand interacts with Fas on activated T lymphocytes some of which recognize paternal antigens. The induction of apoptosis in these T cells prevents them from recognizing and destroying cytotrophoblasts that are invading into the myometrium. A reduced capacity of cytotrophoblasts to invade the spiral arteries, as a consequence of enhanced trophoblast destruction by T cells, results in the development of hypertension and pre-eclampsia.
The Fas gene, known by the gene symbol TNFRSF6, is polymorphic in the promoter region at position –670. A single nucleotide substitution of guanine (TNFRSF6–670*G) for adenine (TNFRSF6–670*A) at this site is associated with decreased Fas production (Pinti et al., 2002; Lai et al., 2003). Therefore, the presence of TNFRSF6–670*G might be associated with a reduced genetic ability to induce apoptosis in maternal lymphocytes and an increased susceptibility to develop pre-eclampsia.
The objective of our study was to determine the association between the TNFRSF6–670 polymorphism and the development of pre-eclampsia.
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Materials and methods |
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Study population
The retrospective study population consisted of 89 normotensive Hungarian women and 38 with pre-eclampsia who delivered singleton pregnancies at Semmelweis University Medical School Hospital between September 2003 and March 2004. The normotensives were randomly selected from a total population of 270 pregnant women. The incidence of pre-eclampsia in this group was 5%. Subsequently, buccal swabs were collected from additional women with pre-eclampsia to increase this sample size. Since the frequency of the Fas gene polymorphism might vary with ethnicity, women from other populations as well as those with multiple gestations or pregnancy-induced diabetes mellitus were excluded. The study was approved by the Committee for Human Rights in Research at the Weill Cornell Medical Center and the corresponding authority at Semmelweis University Medical School.
The diagnosis of pre-eclampsia was made by the attending physician on the basis of a blood pressure of 140/90 mmHg or more after the 20th week of gestation and proteinuria >300 mg/24 h in the collected urine sample. Patients with hypertension present before gestation or having an onset before the 20th week of gestation, and those without proteinuria, were diagnosed as having chronic hypertension or pregnancy-induced hypertension and were not included in the study. IUGR was diagnosed in neonates when their birthweight at delivery was below the 10 percentile value for the given gestational age.
Specimens
Cells from the buccal mucosa were obtained by rotating a cotton swab against the inside of the cheek. Specimens were collected from both mother and her neonate within 1 h after delivery and stored at 4 °C for further testing. Mothers rinsed their mouth with tap water before collection. Shipment to Weill Medical College was by overnight mail at ambient temperature. The specimens were stored at 4 °C until analysed. Clinical data were blinded to those performing the gene polymorphism testing.
TNFRSF6–670 gene polymorphism analysis
The single nucleotide polymorphism at position –670 in the TNFRSF6 gene promoter creates a Mva1 restriction site (Pinti et al., 2002). Cell lysis to release DNA was accomplished by incubation of the buccal cells in a Brij detergent-containing buffer in the presence of proteinase K, as described previously (Genc et al., 2002). Aliquots (15 µl) were diluted to 25 µl with water and added to an equal volume of reaction mixture (10 mmol/l Tris–HCl containing 1.5 mmol/l MgCl2, 50 mmol/l KCl, 0.2 mmol/l each of dATP, dCTP, dGTP and TTP, 1.25 units of Taq DNA polymerase) containing 30 pmol of primer-pairs specific for the polymorphic region: 5'-CTA CCT AAG AGC TAT CTA CCG TTC-3' and 5'-GGC TGT CCA TGT TGT GGC TGC-3' (Pinti et al., 2002). Samples were incubated in a thermal cycler at 94°C for 6 min followed by 30 cycles at 94°C for 30 s, 62°C for 30 s and 72°C for 60 s. This was followed by a single extension cycle at 72°C for 10 min. The amplicons were digested for 5 h at 37°C with Mva1 restriction endonuclease (New England Biolabs, Beverly, MA) and the fragments separated on 3% agarose gels and visualized with ethidium bromide. TNFRSF6–670*A yielded a single 233 base pair band while TNFRSF6–670*G yielded two 189 and 44 base pair bands. Water blanks (negative control) and a DNA sample heterozygous for the two TNFRSF6–670 alleles (positive control) were always analysed in parallel to the test samples. Replicate analyses of selected samples always yielded consistent results.
Statistics
Genotype and allele frequencies were determined by direct counting and then dividing by the number of chromosomes to obtain allele frequency and by the number of women to obtain genotype frequency. Associations between the maternal and the fetal TNFRSF6 genotypes and presence of pre-eclampsia were analysed by the chi-square test, and Fisher's exact test if appropriate. Goodness of fit to Hardy–Weinberg equilibrium was determined by comparing the expected genotype frequencies with the observed values, using the chi-square test. A P value of <0.05 was considered significant. In addition, odds ratios (OR) and confidence intervals (CI) were calculated for each comparison.
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Results |
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Pregnancy outcome
Of the 127 singleton pregnant women enrolled, 38 had pre-eclampsia while 89 normotensive women with uncomplicated pregnancies served as controls. Pregnancy data are summarized in Table I. Patients in the two subgroups were comparable with regard to their mean age. However, mean gestational age and mean birthweights of neonates born to mothers with pre-eclampsia were less than those born to normotensive pregnant women (P<0.01). Pre-eclamptic patients were also more likely to undergo Caesarean delivery than the controls (P<0.01).
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TNFRSF6 gene polymorphism and pre-eclampsia
The association between the TNFRSF6 genotype or allele frequency and the patient diagnosis is shown in Table II. Homozygous TNFRSF6–670*A carriage was detected in 18.4% of pre-eclamptic patients as opposed to 37.1% of controls (P=0.04). In contrast, TNFRSF6–670*G was present in 59.2% of women with pre-eclampsia and 42.1% of controls (P=0.01). The genotype distribution in both patients and controls were in Hardy–Weinberg equilibrium.
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In an attempt to differentiate between early and late onset pre-eclampsia, the patients were divided into those having pre-eclampsia-associated pregnancy termination before (31 patients) and after (7 patients) 37 weeks gestation. Only in the former group there were associations between the homozygosity for TNFRSF6–670*A (P=0.04) and TNFRSF6–670*G carriage (P=0.01) and the pre-eclampsia (Table III).
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TNFRSF6 polymorphism and IUGR
Neonates from 14 (36.8%) pre-eclamptic women and none of the normotensive controls had IUGR. As shown in Table IV, homozygosity for TNFRSF6–670*G was more prevalent in pre-eclamptic women with IUGR babies than in those pre-eclamptics with eutrophic neonates and in normotensive controls without IUGR (P=0.008).
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None of the women with pre-eclampsia who delivered after 37 weeks gestation had a neonate with IUGR. In contrast, IUGR was diagnosed in 14 (45.1%) of neonates from pre-eclamptic mothers who delivered at <37 weeks gestation. The association between the IUGR in these women and the TNFRSF6–670 polymorphism is shown in Table V. TNFRSF6–670*G homozygosity was observed in 57.2% of pre-eclamptics with an IUGR neonate as opposed to only 17.6% of pre-eclamptics without IUGR (P=0.03) and those who delivered a normotensive neonate at <37 weeks (P=0.03). Similarly, TNFRSF6*G carriage was more frequent in pre-eclamptic pregnant women who had a pregnancy termination before 37 weeks gestation and delivered a growth restricted neonate than those who were normotensive and delivered an eutrophic neonate at term (P=0.01).
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Discussion |
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An A>G polymorphism in the TNFRSF6 gene that reduces the expression of Fas is associated with pre-eclampsia at <37 weeks and with IUGR in the corresponding neonates. The results are consistent with the hypothesis that a relative inability to induce apoptosis in maternal lymphocytes results in insufficient trophoblast invasion of the spiral arteries. This, in turn, predisposes to placental ischaemia and vascular endothelial damage (Gerretsen et al., 1981
; Roberts and Redman, 1993; Ness and Roberts, 1996). A recent investigation demonstrated that during the first trimester trophoblast cells have high intracellular concentrations of Fas ligand, which are secreted in microvesicles and induce Fas-mediated apoptosis of maternal T lymphocytes (Abrahams et al., 2004). Thus, the molecular events culminating in pre-eclampsia undoubtedly are initiated prior to detection of clinical symptoms. Although the present investigation was retrospective, the findings strongly suggest that women who are TNFRSF6–670*G homozygous (or negative for TNFRSF6–670*A) are at increased risk for developing pre-eclampsia.
The TNFRFS6–670*G polymorphism was also associated with IUGR in women with pre-eclampsia in whom delivery had to be terminated for hypertension at <37 weeks gestation. Prior studies have noted a relationship between delivery at <37 weeks, IUGR and pre-eclampsia. Birthweights for preterm infants were significantly reduced if the mothers had pre-eclampsia than if they were normotensive (Skjaerven et al., 2000; Xiong et al., 2002). Interestingly, pre-eclampsia was not associated with a decreased birthweight in neonates delivered at term (>37 weeks gestation). Although the number of pre-eclamptic patients in our present study who delivered at >37 weeks were few to reach definitive conclusions, we did not observe an increased rate of TNFRFS6–670*G carriage in this population. Thus, it is reasonable and biologically plausible to hypothesize that the same genetic mechanism responsible for a decrease in Fas-related apoptosis also impairs fetal growth. Additional studies are needed to assess the possible relationship between the TNFRSF6 polymorphism and the delivery of IUGR infants by mothers who do not have pre-eclampsia.
Fetal DNA can be detected in the maternal circulation during pregnancy and the concentration of this DNA is increased in women who will subsequently develop pre-eclampsia (Lo et al., 1999; Cotter et al., 2004). Furthermore, there appears to be an association between the quantity of fetal DNA detected and the risk for pre-eclampsia. The elevated levels of fetal DNA in the maternal circulation might be due to an increased rate of syncytiotrophoblast apoptosis in women with pre-eclampsia and/or with a relative impairment in the ability to induce apoptosis in maternal T cells due to the TNRFSF6 gene polymorphism.
Not all women who were homozygous for TNFRSF6*G had pre-eclampsia. Other factors undoubtedly influence the likelihood of adverse pregnancy events in women carrying this genotype. Additional factors, such as the extent of non-classical HLA antigen expression (Kovats et al., 1990) as well as differential cytokine and matrix metalloproteinase production (Lala and Graham, 1990; Bischof et al., 2000) are also involved in determining the extent of trophoblast invasion. Elucidation of the association between the additional genetic polymorphisms and the pre-eclampsia will undoubtedly lead to a further refinement in the capability to pinpoint women who are truly at risk for early onset pre-eclampsia and associated IUGR. An association between the TNFRSF6–670A>G polymorphism in the firstborn neonate of multi-fetal pregnancies and the preterm premature rupture of the fetal membranes has recently been reported (Kalish et al., 2005). Thus, this Fas polymorphism under appropriate conditions appears to have the potential to also influence other pregnancy-related events.
Validation of the influence of TNFRSF6 polymorphism on development of pre-eclampsia and IUGR by prospective investigations is necessary and would enable clinicians to identify pregnant women at increased risk for development of this disorder and who would benefit from increased screening.
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Acknowledgements |
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This investigation was supported by National Institutes of Health grant HD41676 and M01RR0047 from the Division of Clinical Research, National Center for Research Resources.
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Submitted on October 13, 2004; accepted on January 7, 2005.
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