European Journal of Obstetrics & Gynecology and Reproductive Biology 148 (2010) 144–146
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Polymorphisms of FAS and FAS ligand genes in preeclamptic women Pasquapina Ciarmela a,*, Sonia Boschi b, Enrrico Bloise b, Luca Marozio c, Chiara Benedetto c, Mario Castellucci a, Felice Petraglia b a
Department of Molecular Pathology and Innovative Therapies, Faculty of Medicine, Polytechnic University of Marche, via Tronto 10/a, 60020 Ancona, Italy Obstetrics and Gynecology, Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy c Department of Gynecology and Obstetrics, University of Turin, Turin, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 February 2009 Received in revised form 24 June 2009 Accepted 20 October 2009
Objectives: This study investigated the influence that Fas and Fas ligand gene polymorphisms might have on preeclampsia. The pathogenesis of preeclampsia is still enigmatic and several studies have proposed that it may, in part, be determined by genetic susceptibility. Therefore, the identification of a gene polymorphism associated with an increased risk of preeclampsia might well represent a useful tool in the identification of at risk pregnant women enabling the setup of preventive therapy. Apoptosis has also been implied in the pathogenesis of preeclampsia and since Fas and Fas ligand are the main apoptotic pathway members, they may represent candidate genes involved in the development of preeclampsia. A polymorphism at the 670 position (A–G) in the Fas gene has been found more frequently in Hungarian women with preeclampsia. Study design: The study cohort was a group of 50 women with preeclampsia and 142 healthy control subjects from the general Italian population. They were studied, by RFLP analysis, to validate the role that the 670 G Fas gene polymorphism plays in preeclampsia, and to evaluate the Fas ligand IVS2nt 124 G polymorphism. The Fisher’s exact test was used to compute the statistical difference between groups. Results: The presence of the 670 G Fas gene variant was observed in 42 preeclamptic patients (84%) and 96 members of the general population control group (67.6%) (p = 0.029). Regarding the Fas ligand gene, the IVS2nt 124 G variant was present in 14 preeclamptic patients (28%) and in 47 of the general population control subjects (33.1%) (p = 0.6). Conclusions: The present study validated the hypothesis that the Fas 670 G variant may have an influencing role in preeclampsia. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Fas Fas ligand Preeclampsia Genetic polymorphism Pregnancy
1. Introduction Preeclampsia is a complex disorder with a wide clinical spectrum symptoms and occurs in at least 5–10% of pregnancies affecting seriously both mother and fetus. Its initial hallmark is new-onset gestational hypertension and proteinuria in the latter half of pregnancy, usually resolving only after delivery [1]. Although there are still no well defined clear notions as to the mechanisms underlying this syndrome [2], it has been proposed that uteroplacental ischemia [3], endothelial cell dysfunction [4], exaggerated maternal inflammatory response to deported trophoblast [5,6] as well as apoptosis [7–9] may be possible causes. Apoptosis, or programmed cell death, is a highly organized cell death process essential for organ development, tissue remodel-
* Corresponding author. Tel.: +39 0712206270; fax: +39 0712206087. E-mail address:
[email protected] (P. Ciarmela). 0301-2115/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2009.10.026
ling, immune response, tumour suppression and maintenance of tissue homeostasis [10–12]. This process occurs through two broad pathways: the intrinsic pathway (also known as the mitochondrial pathway) and the extrinsic pathway (also known as the death receptor pathway) [13]. Via the intrinsic pathway it can be triggered by any stimuli that causes oxidative stress, mitochondrial disturbances and/or DNA damage, such as therapeutic cancer agents, hypoxia, ischemia-reperfusion injury and ionizing irradiation. Via the extrinsic pathway, apoptosis is induced by the ligand binding of death receptors. The most important ligand–death receptor system includes TRAIL–TRAIL receptors (including TRAIL-R1, also known as DR4 and TRAIL-R2 also known as DR5), the tumour necrosis factor (TNF)–tumour necrosis factor receptor 1 (TNFR1) and Fas ligand–Fas (also known as CD 95 or Apo 1) [14]. The Fas ligand (CD95/APO-L) was first identified as an inducer of cell death in human cancer and leukaemia cell lines [15] and induces apoptosis by binding to Fas, its cognate receptor. Fas is
P. Ciarmela et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 148 (2010) 144–146
ubiquitously expressed, whereas FasL is principally expressed in defined cell lineages, such as activated T lymphocytes, natural killer (NK) cells and tumour cells, as well as in somatic cells in areas of immune privilege (i.e. the placenta). The binding of FasL to Fas leads to the recruitment of FADD (Fas-associated death domain) forming a complex termed DISC (death-inducing signaling complex), which can bind to initiator caspases (caspase 8 and 10), thereby triggering the activation of caspases 3, 7 and 9, leading to apoptotic events [16]. Sziller et al. [17] reported that the G 670 Fas gene variant was present in preeclamptic (31/38, 81%) Hungarian women more than it was in normotensive (56/89, 62%) women. There is evidence to suggest that preeclampsia may, in part, be determined by genetic susceptibility [1]. The identification of a gene polymorphism associated with an increased risk of preeclampsia could represent a useful tool in the identification of at risk pregnant women and, therefore, allow for the setup of a preventive possible therapy [18]. As the Fas and Fas ligand may represent candidate genes involved in the development of preeclampsia, the presence of a genetic variant might increase susceptibility for the development of the pathology. As population genetic analyses of polymorphisms may differ depending on the ethnic group, it was necessary to carry out a further evaluation of the Fas polymorphism in order to validate this hypothesis. On the other hand, the evaluation of the Fas ligand polymorphism was also needed. Therefore, so as to further evaluate any association the Fas polymorphism and the Fas ligand may have with preeclampsia, we investigated whether the G 670 Fas gene variant and the Fas ligand INV2nt 124 G variant had a differential distribution in the Italian patients with preeclampsia compared to that observed in the general population. 2. Materials and methods 2.1. Patient information Gene polymorphisms for Fas and FasL were analyzed in Caucasian subjects recruited in a previous study on mutational analysis in preeclampsia [19]. Briefly, the study cohort was made up of: (1) women affected by PE: 50 subjects (age range: 31–39 years); (2) normal control samples from the general population: 102 women and 41 men (age range: 35–65 years). Approval was given by the local ethics committee and informed written consent obtained from all subjects, prior to their inclusion in the study. 2.2. Genotyping Genomic DNA was extracted from 3 ml of blood according to the Lahiri and Nurngerger protocol [20]. FAS and FASLG polymorphisms were identified by the PCR-RFLP method, as previously described [21]. The target fragments containing these two polymorphisms were amplified by the primers: 50 -ATAGCTGGGGCTATGCGATT-30 (forward) and 50 -CATTTGACTGGGCTGTCCAT-30 (reverse) for the FAS 670 A > G [22] and 50 -GCAGTTCAGACCTACATGATTAGGAT-30 (forward) and 50 -CCAGATACAGACCTGTTAAATGGGC-30 (reverse) for FASLG IVS2nt 124 A > G [23]. The amplified PCR products were 193 and 230 for 670 A > G and IVS2nt 124 A > G, respectively. The ScrFI and FokI restriction enzymes (New England Biolabs) were used to distinguish the 670 A > G and IVS2nt 124 A > G polymorphisms, respectively, resulting in 136 and 57 bp fragments for the 670 G allele and 180 and 50 bp fragments for the IVS2nt 124 G allele.
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2.3. Statistical analysis The Fisher’s exact test was used to compute the statistical difference between groups and was considered significant when the p value was G polymorphism A RFLP analysis of the FAS 670 A > G was performed in 50 preeclamptic samples, demonstrating the presence of the 670 G in heterozygosis (GA) in 29 patients (58%) and in homozygosis (GG) in 13 (26%) patients. The RFLP analysis of the FAS 670 A > G in the general population controls (142 subjects) evidenced the presence of the 670 G in heterozygosis (GA) in 68 subjects (47.9%) and in homozygosis (GG) in 28 (19.7%) subjects. Therefore, 670 G polymorphism was observed in a total of 42 preeclamptic patients (84%) and in 96 subjects from the general population (67.6%) (p = 0.029). 3.2. Fas ligand IVS2nt 124 A > G polymorphism A RFLP analysis of the FASLG IVS2nt 124 A > G was performed in 50 preeclamptic samples showing the presence of the IVS2nt 124 G in heterozygosis (GA) in 12 patients (24%) and in homozygosis (GG) in 2 (4%) patients. The RFLP analysis of the FASLG IVS2nt 124 A > G in the general population controls (142 subjects) demonstrated the presence of the IVS2nt 124 G in heterozygosis (GA) in 38 subjects (26.8%) and in homozygosis (GG) in 9 (6.3%) subjects. Thus, IVS2nt 124 G polymorphism was observed in a total of 14 preeclamptic patients (28%) and in 47 subjects from the general population (33.1%) (p = 0.6). The genotypic and allelic frequencies of FAS and FasL are summarised in Table 1. There was no statistically significant difference in the polymorphisms distribution between males and females in the general population. 4. Discussion To the best of our knowledge, the data obtained in this study confirmed, for the first time, the association of the 670 G variant in the Fas gene with the preeclampsia in the Italian population. Table 1 Genotypic and allelic frequencies of FAS and FASL among 50 case patients and 142 general population control subjects. Genotype
Preeclampic women (50), No. (%)
Control subjects (142), No. (%)
FAS position 670 AA GA GG GA + GG Allele A Allele G
8 29 13 42 45 55
(16%) (58%) (26%) (84%) (45%) (55%)
46 68 28 96 160 124
(32.4%) (47.9%) (19.7%) (67.6%) (56.3%) (43.7%)
FASLG position IVS2nt AA GA GG GA + GG Allele A Allele G
36 12 2 14 84 16
(72%) (24%) (4%) (28%) (84%) (16%)
95 38 9 47 228 56
(66.9%) (26.8%) (6.3%) (33.1%) (80.3%) (19.7%)
P. Ciarmela et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 148 (2010) 144–146
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Table 2 670 G FAS gene polymorphism in Italian (present work) and Hungarian [17] women with preeclampsia.
Preeclampic women Control subjectsd a b c d
c
Italiana
Hungarianb
84% 67.6%
81% 62%
Italian preeclampic women vs controls, Fisher exact p = 0.029. Hungarian preeclampic women vs controls, Fisher exact p = 0.04. Italian vs Hungarian preeclampic women, Fisher exact p = 0.78. Italian vs Hungarian controls, Fisher exact p = 0.479.
As reported in Table 2, the incidence of the 670 G Fas gene polymorphism in Italian women with preeclampsia did not differ from that of the Hungarian women with preeclampsia, analyzed by Sziller et al. [17]. Genetic origin and the relationship between Hungarians and other European populations have been a source of debate in some reports [25– 27] which are not without discrepancies. However, as Hungarians originate from Asia, they are unique among the other European populations. Since population genetic analyses of polymorphisms may vary according to the ethnic group, the finding that both Italian and Hungarian populations showed the same polymorphism, despite their different origin, is a relevant finding. Although the frequency of this polymorphism may underlie other population-specific differences and the study is somewhat limited by the small size of the sample investigated, the fact that there was a higher prevalence of the Fas 670 G variant in preeclamptic patients in two populations with different genetic origins does offer support for the hypothesis that it contributes to preeclampsia susceptibility. The normal endovascular invasion by trophoblastic cells is impaired in preeclampsia and it has been suggested that abnormal implantation of the trophoblast might be genetically determined [1]. The Fas 670 G variant is associated with a decreased Fas production in activated T lymphocytes [23,28], which may well alter the immune privileged site of the trophoblast and, therefore, contribute to the development of the preeclampsia. Under normal conditions, activated T lymphocytes, some of which recognize paternal antigens, expressing Fas interact with trophoblast expressing the Fas ligand [29]. Therefore, the induction of apoptosis in these T cells makes them unable to recognise and destroy the cytotrophoblasts invading the myometrium and spiral arteries. On the contrary, in the presence of hypertension and preeclampsia, T cells, that express a reduced amount of Fas, have a reduced apoptosis and are, therefore, able to enhance the destruction of the cytotrophoblasts. A reduced amount of invading cytotrophoblasts are unable to carry out an adequate modification of the spiral arteries. Interestingly, Miko et al. have recently observed a lower expression of surface Fas antigen on T cells in patients with preeclampsia, in comparison to healthy pregnant women [30]. The data obtained in this study may represent a first validation step of the influence of the 670 G Fas gene variant on the development of preeclampsia. This knowledge is important not only to further explain the mechanism of pathogenesis, but would also enable clinicians to identify pregnant women at risk for the development of this disorder. Indeed, this study, not only evaluated the Fas 670 G variant, but also the presence of the Fas ligand IVS2nt 124 G variant. There was no evidence that the change from A to G at the nucleotide position 124 of intron 2 was associated with preeclampsia. In conclusion, the research reported herein supports the hypothesis
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