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ORIGINAL RESEARCH |
From the Departments of Obstetrics and Gynecology, Clinical Chemistry, Neurology, and Chromosome and DNA Laboratory of the Division of Diagnostic Services, Kuopio University Hospital, Kuopio, Finland.
Address reprint requests to: Seppo Heinonen, MD, PhD, Department of Obstetrics and Gynecology, Kuopio University Hospital, 70211 Kuopio, Finland; E-mail: seppo.heinonen{at}kuh.fi.
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ABSTRACT |
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 ABSTRACT MATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: The study involved 133 preeclamptic and 115 healthy control pregnant women who were genotyped for the G-455A polymorphism in the ß-fibrinogen gene promoter and for a decamer insertion or deletion polymorphism at position -323 in the factor VII gene promoter. We used 
2 analysis to assess genotype frequency differences between preeclamptic women and controls.
RESULTS: The allelic distribution of the fibrinogen A-455G polymorphism was similar in the two groups, with the frequency of the variant A allele being 18.8% in the preeclampsia group and 20.9% in the control group. We did not find any association between the presence of the factor VII insertion allele and preeclampsia (5.6% versus 6.1%). Accordingly, the genotype distribution of the fibrinogen G-455A and factor VII polymorphisms in the preeclamptic and control groups was similar (P = .852 and P = .308).
CONCLUSION: The G-455A polymorphism of the fibrinogen gene promoter and the decamer insertion or deletion polymorphism of the factor VII gene promoter are unlikely to be major genetic predisposing factors for preeclampsia in subjects from eastern Finland.
Hemostatic abnormalities associated with an increased risk of thrombosis are reported to be more common among women with obstetric complications than among women with normal pregnancies.1–3 Thrombotic features of placental vascular lesions suggest that thrombophilia, hypofibrinolysis, or both reduce uteroplacental blood flow by means of intervillous or spiral artery thrombosis and result in hypoxia. These changes make miscarriage, preeclampsia, fetal growth restriction, abruptio placentae, and stillbirth more likely than they would be otherwise.1,4 The hemostatic abnormalities most often considered include deficiency of protein C, protein S, and antithrombin III; the presence of anticardiolipin antibodies; activated protein C resistance; and mutations in the genes encoding methylenetetrahydrofolate reductase and prothrombin.5–10
Polymorphisms in the genes encoding fibrinogen and factor VII have been identified in patients with coronary artery disease.11,12 Tybjaerg-Hansen et al11 found that the (G-455 to A) mutation in the promoter region of the ß-fibrinogen gene is associated with an increase in plasma fibrinogen in both genders in the general population, but this increase did not appear to cause ischemic heart disease. The study of Girelli et al12 suggested that the factor VII genotypes that are associated with the lowest levels of activated factor VII have a role in protection against myocardial infarction. The contribution of these genes to the pathophysiology of preeclampsia has not been assessed previously. The human factor VII gene is located on the long arm of chromosome 13. The factor VII polymorphism involves a 10-bp insertion at position -323 in the 5' promoter region of the factor VII gene; allele D corresponds to the absence of the decamer, and allele I refers to its insertion. Subjects with the II genotype have been shown to have a 66% reduction in activated factor VII activity, and a similar, albeit less pronounced, effect has been seen among heterozygous mutation carriers.12 The gene encoding the human beta-fibrinogen is located on the long arm of chromosome 4. A common mutation, a G-to-A transition at nucleotide position -455 within the promoter of the fibrinogen gene, is associated with elevated plasma fibrinogen levels.11
On the basis of this information linking the pathogenesis of preeclampsia to hemostatic abnormalities, a candidate gene approach was applied in the current study. We hypothesized that the G-455A mutation of the fibrinogen promoter region and/or the insertion or deletion of the factor VII promoter could be important risk factors for preeclampsia.
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MATERIAL AND METHODS |
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Information was collected retrospectively in connection with 133 preeclamptic pregnancies of primiparous women and 115 control women with no history of preeclampsia who delivered at Kuopio University Hospital between January 1994 and December 1998. Using the birth registry at Kuopio University Hospital, we sought preeclamptic patients, called them back, and at that time asked them to give informed consent for the present study. During the same time, we collected blood samples from controls who had given birth in the same hospital after uncomplicated pregnancies and who had at least two normal pregnancies, including the current one. Blood from controls was drawn for DNA analysis at enrollment.
To ensure homogeneity of the genetic background, the controls, who originated from a regional population and had no clinical signs of preeclampsia, were enrolled by random selection in this case-control study.
Preeclampsia was defined as the development of hypertension and new-onset proteinuria (>300 mg of urinary protein in 24 hours) in women with no proteinuria at baseline. Hypertension was defined according to current guidelines that accept 140 and 90 mm Hg of systolic and diastolic pressure, respectively, or higher, as hypertension, when measured on two consecutive occasions at least 24 hours apart.13 Women with chronic hypertension were excluded from the study.
DNA was extracted from peripheral blood lymphocytes using a standard phenol-chloroform extraction method. The G-455A mutation site within the promoter of the ß-fibrinogen gene was amplified by polymerase chain reaction (PCR), and the amplified DNA fragment (1301 bp) was digested with HaeIII restriction enzyme.14 Digestion products were subjected to agarose gel electrophoresis. The PCR product was sequenced using an ABI PRISM 310 genetic analyzer (Perkin Elmer, Foster City, CA). The decamer insertion or deletion polymorphism at position -323 in the promoter region of the factor VII gene was amplified using PCR primers (the forward PCR primer being fluorescently labeled with 4,7,2',7'-tetrachloro-6-carboxyfluorescein) and conditions previously described.15 The sizes of the PCR products were determined using the ABI PRISM 310 genetic analyzer (Perkin Elmer).
Two-tailed pooled t tests were used to analyze continuous variables. Statistical analyses for comparing individual genotype frequencies as well as pooled genotype frequencies were carried out using Pearson 2 test (two-sided asymptotic P values) with SPSS 9.0 software (SPSS Inc., Chicago, IL), and the level of statistical significance was defined as P < .05. Hardy-Weinberg distribution of genotypes in the preeclamptic and control groups was assessed by using the Associate program 2.31 (Hinxton, Cambridge, UK). Sample size and power determinations were performed using nQuery Advisor 4.0 software (Statistical Solutions, Saugus, MA). We used frequency data from the ß-fibrinogen and factor VII promoter genotypes to calculate the necessary sample sizes to achieve a power of 80% and an 
of 0.05.
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RESULTS |
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. All preeclamptic women were nulliparas, whereas controls had two or more deliveries. However, in the control group we also used the case records of their first pregnancies for comparison to exclude the confounding effect of parity on pregnancy outcome. On average, deliveries occurred 5 weeks earlier in preeclamptic women than in controls. The mean (± standard deviation [SD]) gestational age at the development of preeclampsia was 31.7 ± 3.5 weeks. When only the first-degree relatives of the index cases were taken into account, a positive family history was reported in 30 affected women, who had six affected sisters and 28 affected mothers.
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. The distribution of G and A alleles was equal in preeclamptic and unaffected women, as was the distribution of genotypes (P = .852). Genotypes were found to be in Hardy-Weinberg equilibrium in the study and control groups.
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). The distribution of insertion and deletion alleles was equal in preeclamptic and unaffected women, as was the distribution of genotypes (P = .308). Genotypes were found to be in Hardy-Weinberg equilibrium in both the study and control groups. However, almost-significant deviation from Hardy-Weinberg equilibrium was noted in the control group, which was considered to have originated from the low number of ID and II genotypes in the analysis and not from genotyping errors or population stratification.
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DISCUSSION |
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The Finnish population is considered a genetic isolate; therefore, it is ideal for genetic association studies.18 In this relatively homogeneous population, any other polymorphism of the genes under consideration is likely to be in linkage disequilibrium with those investigated in this study.
We conclude that the polymorphisms of the fibrinogen gene and the factor VII gene are unlikely to be major genetic predisposing factors for preeclampsia in subjects from eastern Finland. However, the possibility that alternative mutations or polymorphisms of the factor VII gene or the fibrinogen gene might segregate with preeclampsia in a population with a different ethnic background cannot be ruled out. Furthermore, our study size did not permit us to analyze data separately for preeclampsia of early and late onset.
Familial genetic predisposition can be investigated in association studies, and the results of the present study suggest that there is an equilibrium state between factor VII and fibrinogen genotypes and alleles in preeclamptic women and women in the control group. We did not find a significant relationship between the factor VII or fibrinogen polymorphism and preeclampsia; however, the limitation of our study is its relatively small sample size, which theoretically increases the likelihood of a type II error. Power analysis showed that to detect a difference for fibrinogen and factor VII allele frequencies with a power of 80% and an alpha of .05 we would have needed to enroll 2898 and 34,583 preeclamptic and control women in each group, respectively. These figures suggest that it is unlikely that our findings are false negatives.
Unlike many other diseases, genetic studies on pre-eclampsia have been focused on finding and testing candidate genes. There are three main reasons for this. First, localizing preeclampsia genes by means of classic systematic linkage studies has been difficult because of the heterogeneity of the etiology of the disease, uncertainty as to the type of inheritance, and nonexistent male phenotype. Second, it has been possible to name several attractive candidate genes, such as the genes affecting vascular relaxation and immune responses. Third, studies on hemostatic variables in maternal serum have been hampered by activated coagulation in the placental vessels in preeclampsia, as initially evidenced by abnormal factor VIII consumption.19,20 One of the strengths of the candidate gene approach in the current study is the existence of the Finnish closed heritage, which is genetically relatively homogeneous. Collectively, this report presents no evidence indicating that the decamer insertion or deletion polymorphism in the factor VII gene promoter or the G-455A polymorphism in the fibrinogen gene promoter has an association with preeclampsia, and thus other genes should be targets for further studies in the field of preeclampsia research.
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Footnotes |
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Received December 26, 2001. Received in revised form March 8, 2002. Accepted March 21, 2002.
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REFERENCES |
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2. van Pampus MG, Dekker GA, Wolf H, Huijgens PC, Koopman MM, von Blomberg BM, et al. High prevalence of hemostatic abnormalities in women with a history of severe preeclampsia. Am J Obstet Gynecol 1999;180: 1146–50.[Medline]
3. Kupferminc MJ, Eldor A, Steinman N, Many A, Bar-Am A, Jaffa A, et al. Increased frequency of genetic thrombophilia in women with complications of pregnancy. N Engl J Med 1999;340:9–13.
4. Arias F, Romero R, Joist H, Kraus FT. Thrombophilia: A mechanism of disease in women with adverse pregnancy outcome and thrombotic lesions in the placenta. J Maternal Fetal Med 1998;7:277–86.
5. Yasuda M, Takakuwa K, Tokunaga A, Tanaka K. Prospective studies of the association between anticardiolipin antibody and outcome of pregnancy. Obstet Gynecol 1995;86:555–9.[Abstract]
6. de Vries JI, Dekker GA, Huijgens PC, Jakobs C, Blomberg BM, van Geijn HP. Hyperhomocysteinaemia and protein S deficiency in complicated pregnancies. Br J Obstet Gynaecol 1997;104:1248–54.[Medline]
7. Kupferminc MJ, Fait G, Many A, Gordon D, Eldor A, Lessing JB. Severe preeclampsia and high frequency of genetic thrombophilic mutations. Obstet Gynecol 2000; 96:45–9.
8. Wang J, Trudinger BJ, Duarte N, Wilcken DE, Wang XL. Elevated circulating homocyst(e)ine levels in placental vascular disease and associated pre-eclampsia. Br J Obstet Gynaecol 2000;107:935–8.
9. Alfirevic Z, Mousa HA, Martlew V, Briscoe L, Perez-Casal M, Toh CH. Postnatal screening for thrombophilia in women with severe pregnancy complications. Obstet Gynecol 2001;97:753–9.
10. Livingston JC, Barton JR, Park V, Haddad B, Phillips O, Sibai BM. Maternal and fetal inherited thrombophilias are not related to the development of severe preeclampsia. Am J Obstet Gynecol 2001;185:153–7.[Medline]
11. Tybjaerg-Hansen A, Agerholm-Larsen B, Humphries SE, Abildgaard S, Schnohr P, Nordestgaard BG. A common mutation (G-455 
A) in the beta-fibrinogen promoter is an independent predictor of plasma fibrinogen, but not of ischemic heart disease. A study of 9,127 individuals based on the Copenhagen City Heart Study. J Clin Invest 1997; 99:3034–9.[Medline]
12. Girelli D, Russo C, Ferraresi P, Olivieri O, Pinotti M, Friso S, et al. Polymorphisms in the factor VII gene and the risk of myocardial infarction in patients with coronary artery disease. N Engl J Med 2000;343:774–80.
13. Report of the National High Blood Pressure Education Program Working Group on high blood pressure in pregnancy. Am J Obstet Gynecol 2000;183:S1–S22.
14. Thomas AE, Green FR, Kelleher CH, Wilkes HC, Brennan PJ, Meade TW, et al. Variation in the promoter region of the beta fibrinogen gene is associated with plasma fibrinogen levels in smokers and non-smokers. Thromb Haemost 1991;65:487–90.[Medline]
15. Bernardi F, Marchetti G, Pinotti M, Arcieri P, Baroncini C, Papacchini M, et al. Factor VII gene polymorphisms contribute about one third of the factor VII level variation in plasma. Arterioscl Thromb Vascular Biol 1996;16:72–6.
16. Arngrimsson R, Björnsson H, Geirsson R. Analysis of different inheritance patterns in preeclampsia/eclampsia syndrome. Hypertens Pregnancy 1995;14:27–38.
17. Cincotta RB, Brennecke SP. Family history of pre-eclampsia as a predictor for pre-eclampsia in primigravidas. Int J Gynaecol Obstet 1998;60:23–7.[Medline]
18. Peltonen L, Jalanko A, Varilo T. Molecular genetics of the Finnish disease heritage. Hum Mol Genet 1999;8: 1913–23.
19. Redman CW, Denson KW, Beilin LJ, Bolton FG, Stirrat GM. Factor-VIII consumption in pre-eclampsia. Lancet 1977;2:1249–52.[Medline]
20. Schjetlein R, Abdelnoor M, Haugen G, Husby H, Sandset PM, Wisloff F. Hemostatic variables as independent predictors for fetal growth retardation in preeclampsia. Acta Obstet Gynecol Scand 1999;78:191–7.[Medline]
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