OBJECTIVE: To estimate whether an A>G polymorphism at position −670 in the gene coding for Fas (gene symbol TNFRSF6) is associated with hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome.
METHODS: In a retrospective study, buccal swabs from 81 women with the complete form of HELLP syndrome and 83 normotensive control women with uncomplicated full-term pregnancy, and 110 of their neonates, were analyzed for the presence of the TNFRSF6–670 polymorphism. Investigators were blinded to clinical outcomes.
RESULTS: Pregnant women heterozygous for the TNFRSF6–670 genotype were more likely than those homozygous for TNFRSF6–670*A allele to have HELLP syndrome (P = .01; odds ratio 2.7, 95% confidence interval 1.2–5.9). Moreover, patients with homozygous carriage of the TNFRSF6–670*G allele were more likely than those homozygous for the wild type of the Fas gene (TNFRSF6–670*A/A) to have HELLP syndrome (P = .006; odds ratio 4.0, 95% confidence interval 1.7–9.8). In contrast, TNFRSF6–670 genotype distribution of neonates born to mothers with HELLP syndrome was not statistically different from that found in neonates born to healthy pregnant women (P = .4). In patients with HELLP syndrome, no association between TNFRSF6 genotype distribution and severity of hemolysis, platelet counts or liver enzymes levels was noted.
CONCLUSION: A single A>G nucleotide substitution at position −670 in the maternal but not neonatal TNFRSF6 gene coding for Fas is associated with a higher risk for HELLP syndrome.
LEVEL OF EVIDENCE: II-2
A single A&#x003E;G nucleotide substitution at position -670 in the maternal but not neonatal TNFRSF6 gene coding for Fas is associated with a higher risk of HELLP syndrome.
From the 1First Department of Obstetrics and Gynecology, Semmelweis University Faculty of Medicine, Budapest, Hungary; and 2Division of Immunology and Infectious Diseases, Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, New York, New York.
Supported in part by Ignácz Semmelweis Foundation, and National Institutes of Health grant HD 41676.
Corresponding author: István Sziller, MD, First Department of Obstetrics and Gynecology, Semmelweis University, Faculty of Medicine, Baross utca 27, H-1088 Budapest, Hungary; e-mail: firstname.lastname@example.org.
The term hemolysis, elevated liver enzymes and low platelet counts (HELLP) syndrome was introduced to describe a subset of pregnant women with severe preeclampsia who develop multiorgan dysfunction at some stage during their pregnancy or after delivery.1,2 HELLP syndrome is associated with significant maternal and perinatal morbidity and mortality affecting 1 in 400 pregnant women.3–5 Although neonatal morbidity and mortality is mainly a consequence of prematurity and low birth weight, maternal morbidity is largely due to significant hematologic changes, liver disease, and high blood pressure.
The cause of preeclampsia with or without HELLP syndrome remains unknown. Histologic studies of placental bed biopsies from women with severe preeclampsia have shown inadequate invasion of the uterine wall blood vessels by fetal extravillous trophoblast.6 Preeclampsia has also been attributed to a breakdown in maternal tolerance to the fetal semiallograft.7 It is well documented that the Fas-Fas ligand (FasL) system plays an important role in the regulation of both processes.8,9 Fas and Fas L are classical transmembrane proteins that belong to the tumor necrosis factor receptor super family (TNFRSF) of proteins. Fas is expressed by activated lymphocytes and trophoblasts, and delivers a signal to induce apoptosis in these cells.10 Expression of FasL, on the other hand, is limited to certain leukocytes and tissues with immune privilege, including the human trophoblast throughout gestation.11 In successful pregnancies, binding of trophoblast-associated FasL to Fas-expressing activated maternal T lymphocytes that invade the trophoblast during implantation induces apoptosis in Fas-bearing maternal T cells, allowing fetal trophoblast to invade into the myometrium while escaping immune recognition. The Fas-expressing invading trophoblasts also might undergo apoptosis from FasL-expressing maternal T cells, limiting the extent of myometrial invasion.11 Disturbed Fas-mediated apoptosis is involved in the pathogenesis of preeclampsia,12 HELLP syndrome,13 liver diseases with14 and without HELLP syndrome,15 and cytopenias.16
The importance of the Fas gene (gene symbol TNFRSF6) has received recent attention due to its essential role in the regulation of the immune system. The Fas gene is polymorphic at 2 sites, in the silencer at nucleotide position −1377, and in the enhancer region at nucleotide position −670.17 A single nucleotide substitution of adenine (TNFRSF6*A) for guanine (TNFRSF6*G) at position −670 was shown to result in decreased Fas production and altered function of the Fas-FasL system.18 Maternal homozygosity for TNFRSF6*G allele was recently associated with an elevated risk for early onset preeclampsia and intrauterine growth restriction.19 The aim of the present study was to estimate whether a polymorphism in the Fas receptor gene at position −670 in the maternal and fetal genome was associated with the more pronounced HELLP form of preeclampsia.
MATERIALS AND METHODS
Between September 2003 and February 2005, 35 consecutive Hungarian pregnant women with antenatally diagnosed complete form of HELLP syndrome and their neonates who delivered at Semmelweis University Medical School were enrolled and tested for TNFRSF6 genotype. In addition, 46 consecutive Hungarian HELLP patients who attended for annual postpartum medical follow-up visit during the study period and delivered at the same department between January 1998 and August 2003 were also enrolled and tested. Thus, the total number of patients with HELLP syndrome was 81. During the study period, 15 patients had an incomplete form of HELLP syndrome with varying absence of hemolysis, thrombocytopenia, or elevated liver enzymes. Furthermore, 3 patients had HELLP syndrome developed in the postpartum period only. No patients from the latter 2 subgroups were considered as candidates for enrollment.
The control group consisted of 83 consecutive Hungarian full-term pregnant women and their neonates who delivered at the same department between November and December 2004. Enrollment into the control group was offered only for those with an uncomplicated singleton term pregnancy without pregnancy-related medical complication and delivery of a healthy neonate.
Because the frequency of the TNFRSF6 gene polymorphisms might vary with ethnicity, women from other populations as well as those with pregnancy-induced medical complications were excluded. Each participant gave informed consent, and the study was approved by the Committee for Human Rights in Research at Semmelweis University Medical School and the corresponding authority at the Weill Cornell Medical Center.
The diagnosis of HELLP syndrome was made by the presence of thrombocytopenia (below 150,000 cells/μL), evidence of hepatic dysfunction (increased aspartate aminotransferase level of > 70 IU/L, increased alanine aminotransferase level of > 70 IU/L, or both, with increased lactate dehydrogenase level of > 600 IU/L), and evidence of hemolysis (increased serum bilirubin level of > 1.2 mg/dL and increased lactate dehydrogenase level of > 600 IU/L). In this study, all patients with HELLP syndrome had a complete form of the disorder, including hemolysis, elevated liver enzymes, and thrombocytopenia diagnosed antenatally. Pregnant women who had partial HELLP syndrome or those who had onset of the disorder in the postpartum period were not enrolled into the study. Control subjects comprised normotensive women with healthy pregnancies, term deliveries, and a healthy neonate.
Cells from the buccal mucosa were obtained by rotating a cotton swab against the inside of the cheek. Specimens were collected in mother-infant pairs within 1 hour after delivery and stored at 4°C for further testing. Clinical data were blinded to those performing the gene polymorphism testing.
The single A>G nucleotide polymorphism at position −670 in the TNFRSF6 gene promoter creates a Mva1 restriction site. 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.17,20 Aliquots (15 μL) were diluted to 25 μL with H2O and added to an equal volume of reaction mixture (10 mM Tris-HCl containing 1.5 mM MgCl2, 50 mM KCl, 0.2 mM 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′. Samples were incubated in a thermal cycler at 94°C for 6 minutes followed by 30 cycles of 94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 60 seconds. This was followed by a single extension cycle of 72°C for 10 minutes. The amplicons were digested for 5 hours at 37°C with Mva1restriction endonuclease (New England Biolabs, Beverly, MA) and the fragments separated on 3% agarose gels and visualized with ethidium bromide. The TNFRSF6–670*A yielded a single 233 base pair band whereas TNFRSF6–670*G yielded 2 189 and 44 base pair bands. Water blanks (negative control) and a DNA sample heterozygous for the 2 TNFRSF6–670 alleles (positive control) were always analyzed in parallel to the test samples. Replicate analyses of selected samples always yielded consistent results.
Measures with continuous distribution were compared using the Student t test. 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 maternal and fetal TNFRSF6 genotypes and presence of HELLP syndrome were analyzed by the χ2 test. Goodness of fit to Hardy-Weinberg equilibrium was determined by comparing the expected genotype frequencies with the observed values, using χ2.21 A P value less than .05 was considered significant. In addition, odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for comparisons.
Demographic data and obstetric outcome of the study population is summarized in Table 1. Cases and controls were comparable with regard to their mean age and proportion of patients with their first pregnancy. However, mean gestational age of case patients was 7 weeks less compared with healthy pregnant women (P < .01), and the mean birth weight of neonates born to mothers with HELLP syndrome was 1,800 g less than those born to control pregnant women (P < .01). All but 4 (95.1%) patients in the HELLP syndrome group underwent cesarean delivery compared with 21.6% of control patients (P < .001).
The association between TNFRSF6 genotype distribution and patient diagnosis is shown in Table 2. Pregnant women heterozygous for the TNFRSF6–670 genotype were more likely than TNFRSF6–670*A homozygotes to have HELLP syndrome (P = .01; OR 2.7, 95% CI 1.2–5.9). Moreover, patients with homozygous carriage of the TNFRSF6–670*G allele were more likely than those homozygous for the wild type of the Fas gene (TNFRSF6–670*A) to have HELLP syndrome (P = .006; OR 4.0, 95% CI 1.7–9.8). The genotype distribution in both patients and controls was in Hardy-Weinberg equilibrium. In patients with HELLP syndrome, no associations between TNFRSF6 genotype and severity of hemolysis, platelet counts, or liver enzyme levels were noted.
Overall, there were 110 neonatal buccal samples available to be tested for TNRFSF6–670 gene polymorphism (Table 3). Of these neonates, 35 were born to mothers with HELLP syndrome, and 75 to mothers belonging into the control group. Neonates born to mothers with HELLP syndrome were more likely to be either heterozygotes for the TNFRSF6–670 alleles or homozygous for TNFRSF6–670*G allele than those born to healthy pregnant women, but these differences did not reach the level of statistical significance (overall P = .4; OR 1.2, 5% CI 0.4–3.1, and OR 2.0, 95% CI 0.7–5.6, respectively).
In this study, a single A>G nucleotide substitution at position −670 in the maternal but not neonatal TNFRSF6 gene coding for Fas was associated with a higher risk for HELLP syndrome. These data expand on our previous observations that TNFRSF6*G homozygosity in pregnant women increases the risk for early onset preeclampsia and intrauterine growth restriction.19 The TNFRSF6–670 polymorphism has previously been linked to decreased expression of Fas on maternal cells.18 Therefore, we hypothesize that decreased apoptotic potential of maternal T cells due to polymorphism in the Fas gene at −670 contributes to a prolonged capacity of maternal lymphocytes to recognize and destroy fetal trophoblasts during invasion into the uterine wall and spiral arteries. Resulting incomplete implantation in turn would initiate the preeclampsia and HELLP syndrome pathway. Because not only activated maternal T lymphocytes but also spiral artery endothelial and smooth muscle cells express Fas,9 and secretion of FasL by trophoblasts is demonstrated as early as the first trimester,22 reduced sensitivity of blood vessel cells to apoptosis due to maternal TNFRSF6*G homozygosity might be an additional contributor to the onset of the preeclampsia pathway.
Our data are consistent with previous research of other authors who demonstrated disturbed Fas-mediated apoptosis in patients with preeclampsia and HELLP syndrome by either measurement of Fas and FasL in maternal serum, or their expression on maternal and fetal lymphocytes or in trophoblast cells.12,13,23 Kuntz et al24 demonstrated disturbed function of Fas-mediated apoptosis in patients with pregnancy-related hypertension by elevated FasL concentrations in maternal and cord blood sera and by decreased expression of Fas on maternal leukocytes in affected pregnant women. The authors implicated an intrinsically low Fas expression in these patients, which is in accordance with our present data. Decreased Fas expression of maternal lymphocytes due to TNFRSF6–670*G homozygosity, if associated with resistance of these cells to apoptosis, might allow their evasion of normal immune mechanisms and thus could potentially be an inciting factor for the development of pregnancy-related disease. This possibility is supported by observations in Fas-deficient lpr mice, in which a similar mechanism contributes to the development of autoimmune phenomena.25
HELLP syndrome is the most severe form of preeclampsia, involving significant cellular damage in the liver and peripheral platelets and erythrocytes. The role of Fas-mediated apoptosis in the homeostasis and pathophysiology of liver diseases and in cytopenias is well documented.15,16,26 Liver cells constitutively express Fas and are sensitive to FasL.15 In HELLP syndrome, placenta-derived circulating FasL was shown to be responsible for the typical hepatocyte destruction.14 Our observations expand on these data and emphasize that abnormal function of the Fas gene might also be associated with cytopenias and liver damage and lead to the development of the typical dysfunctions in at least a subset of patients with HELLP syndrome.
In our study, possible fetal contribution to the onset of HELLP syndrome was evaluated by analysis of the neonatal TNFRSF6 genotype at position −670. In a recent study, Allaire et al27 investigated placental apoptosis and demonstrated increased trophoblast apoptosis in placentas and differential expression of Fas and FasL on trophoblast cells from women with preeclampsia. Alteration in the balance of mutual induction of apoptosis by maternal and fetal cells, as suggested by these authors, might affect diseases associated with abnormal placentation, including preeclampsia and fetal growth restriction. Although pregnancies of mothers with a neonate homozygous for the TNFRSF6–670*G allele were more likely to be complicated by HELLP syndrome than that of the mothers with a TNFRSF6–670*A homozygous neonate, statistical analysis did not support a significant association between neonatal TNFRSF6 genotype and maternal HELLP syndrome.
The clinical significance of the association between maternal carriage of TNFRSF6*G and HELLP syndrome seen in our study remains to be determined. A single nucleotide polymorphism in the gene coding one of the apoptosis-related proteins is probably not the single underlying cause for the onset of HELLP syndrome. This is evidenced by the 16.1% frequency of patients with HELLP syndrome who were homozygous for TNFRSF6*A allele. Focusing on one protein in a complex mechanism involving multiple inducers and inhibitors of apoptosis might lead to incomplete results. In addition, there were a few limitations to the present study. Polymorphism in the Fas gene at nucleotide position −1377 was not looked for, because the authors did not find data in the literature on the consequences of a G>A substitution at this locus. Furthermore, 3 patients who developed HELLP syndrome postpartum only were not considered for enrollment, and thus their TNFRSF6 genotype was not known to the investigators. Also, patients with an incomplete form of HELLP syndrome (partial HELLP) were not tested, in an attempt to adopt a strict definition of the disease. Moreover, 46 patients with a recent history of HELLP syndrome were recruited from attendants of an outpatient clinic serving their postpartum follow-up. Although patients were enrolled consecutively, sociodemographic or educational characteristics of attendants might have biased the composition of these women. Subsequent studies enrolling more patients and evaluating Fas and FasL polymorphisms along with measurement of serum Fas and FasL levels in mothers and neonates, as well as expression of these proteins on maternal and fetal cells, might provide a more detailed insight into the relationship between Fas-mediated apoptosis and HELLP syndrome.
1. Weinstein L. Syndrome of hemolysis, elevated liver enzymes, and low platelet count: a severe consequence of hypertension in pregnancy. Am J Obstet Gynecol 1982;142:159–67.
2. Walker JJ. Pre-clampsia. Lancet 2000;356:1260–5.
3. Sibai BM, Taslimi MM, el-Nazer A, Amon E, Mabie BC, Ryan GM. Maternal-perinatal outcome associated with the syndrome of hemolysis, elevated liver enzymes, and low platelets in severe preeclampsia-eclampsia. Am J Obstet Gynecol 1986;155:501–9.
4. Martin JN Jr., Blake PG, Perry KG Jr., McCaul JF, Hess LW, Martin RW. The natural history of HELLP syndrome: patterns of disease progression and regression. Am J Obstet Gynecol 1991;164:1500–9.
5. Audibert F, Friedman SA, Frangieh AY, Sibai BM. Clinical utility of strict diagnostic criteria for the HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome. Am J Obstet Gynecol 1996;175:460–4.
6. Brosens JJ, Pijnenborg R, Brosens IA. The myometrial junctional zone spiral arteries in normal and abnormal pregnancies: a review of the literature. Am J Obstet Gynecol 2002;187:1416–23.
7. Dekker GA, Sibai BM. Etiology and pathogenesis of preeclampsia: current concepts. Am J Obstet Gynecol 1998;179:1359–75.
8. Kabelitz D. Apoptosis, graft rejection, and transplantation tolerance. Transplantation 1998;65:869–75.
9. Ashton SV, Whitley GS, Dash PR, Wareing M, Crocker IP, Baker PN, et al. Uterine spiral artery remodeling involves endothelial apoptosis induced by extravillous trophoblasts through Fas/FasL interactions. Arterioscler Thromb Vasc Biol 2005;25:102–8.
10. Bamberger AM, Schulte HM, Thuneke I, Edmann I, Bamberger CM, Asa SL. Expression of apoptosis-inducing Fas ligand (FasL) in human first and third trimester placenta and choriocarcinoma cells. J Clin Endocrinol Metab 1997;82:3173–5.
11. Griffith TS, Brunner T, Fletcher SM, Green DR, Ferguson TA. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 1995;270:1189–92.
12. Hsu CD, Harirah H, Basherra H, Mor G. Serum soluble Fas levels in preeclampsia. Obstet Gynecol 2001;97:530–2.
13. Harirah H, Donia SE, Hsu CD. Serum soluble Fas in the syndrome of hemolysis, elevated liver enzymes, and low platelets. Obstet Gynecol 2001;98:295–8.
14. Strand S, Strand D, Seufert R, Mann A, Lotz J, Blessing M, et al. Placenta-derived CD95 ligand causes liver damage in hemolysis, elevated liver enzymes, and low platelet count syndrome. Gastroenterology 2004;126:849–58.
15. Galle PR, Hofmann WJ, Walczak H, Schaller H, Otto G, Stremmel W, et al. Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage. J Exp Med 1995;182:1223–30.
16. Liu JH, Wei S, Lamy T, Epling-Burnette PK, Starkebaum G, Djeu JY, et al. Chronic neutropenia mediated by Fas ligand. Blood 2000;95:3219–22.
17. Huang QR, Morris D, Manolios N. Identification and characterisation of polymorphisms in the promoter region of the human Apo-1/fas (CD95) gene. Mol Immunol 1997;34:577–82.
18. Pinti M, Troiano L, Nasi M, Moretti L, Monterastelli E, Mazzacani A, et al. Genetic polymorphisms of Fas (CD95) and FasL (CD178) in human longevity: studies on centenarians. Cell Death Differ 2002;9:431–8.
19. Sziller I, Nguyen D, Halmos A, Hupuczi P, Papp Z, Witkin SS. An A > G polymorphism at position −670 in the Fas (TNFRSF6) gene in pregnant women with pre-eclampsia and intrauterine growth restriction. Mol Hum Reprod 2005;11:207–10.
20. Genc MR, Gerber S, Nesin M, Witkin SS. Polymorphism in the interleukin-1 gene complex and spontaneous preterm delivery. Am J Obstet Gynecol 2002;187:157–63.
21. Chakravarti A. Population genetics—making sense out of sequence. Nat Genet 1999;21:56–60.
22. Abrahams VM, Straszewski-Chavez SL, Guller S, Mor G. First trimester extravillous trophoblast cells secrete Fas L which induces immune cell apoptosis. Mol Hum Reprod 2004;10:55–63.
23. Darmochwal-Kolarz D, Leszczynska-Gorzelak B, Rolinski J, Oleszczuk J. The expression and concentrations of Fas/APO-1 (CD95) antigen in patients with severe pre-eclampsia. J Reprod Immunol 2001;49:153–64.
24. Kuntz TR, Christensen RD, Stegner J, Duff P, Koenig JM. Fas and Fas ligand expression in maternal blood and in umbilical cord blood in preeclampsia. Pediatr Res 2001;50:743–9.
25. Singer GG, Carrera AC, Marshak-Rothstein A, Martinez C, Abbas AK. Apoptosis, Fas and systemic autoimmunity: the MRL-lpr/lpr model. Curr Opin Immunol 1994;6:913–20.
26. Faubion WA, Gores GJ. Death receptors in liver biology and pathobiology. Hepatology 1999;29:1–4.
27. Allaire AD, Ballenger KA, Wells SR, McMahon MJ, Lessey BA. Placental apoptosis in preeclampsia. Obstet Gynecol 2000;96:271–6.
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