Mol. Hum. Reprod. Advance Access originally published online on May 18, 2005
Molecular Human Reproduction 2005 11(6):437-440; doi:10.1093/molehr/gah182
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Tumour necrosis factor-
gene haplotype is associated with pre-eclampsia
1Departments of Obstetrics and Gynaecology, 2Neurology and Neuroscience and 3Medicine, University of Kuopio, Kuopio, Finland
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Kuopio University Hospital, 70211 Kuopio, Finland. Email: seppo.heinonen{at}kuh.fi
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Abstract |
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We determined whether polymorphisms in the promoter region of the tumour necrosis factor alpha (TNF-
) gene contributes to differences in susceptibility to develop pre-eclampsia. The study involved 133 pre-eclamptic and 115 healthy pregnant women who were genotyped for the G-308A polymorphism of the TNF- gene. The frequency of the G-308A allele was more common in the pre-eclampsia group than among the controls (P=0.046), giving an odds ratio of 0.57 (95% CI: 0.32–0.99), but there were no differences in the genotype distribution. The data from the G-308A polymorphism was combined with the previously published genotype and allele data from the C-850T polymorphism of the TNF- gene, and used to assess a haplotype estimation analysis. Estimated overall pair of loci haplotype frequencies differed significantly between the groups (P=0.023±0.004). In the single haplotype association analysis, the haplotype C-A versus others was over-represented in the pre-eclampsia group (P=0.041±0.003), whereas the haplotype T-G versus others was less common in the pre-eclampsia group (P=0.035±0.003), compared with the controls. In conclusion, the polymorphisms of the TNF- gene showed a significant haplotype association with susceptibility to pre-eclampsia in the Finnish population. Key words: tumour necrosis factor alpha/polymorphism/pre-eclampsia/haplotype
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Tumour necrosis factor alpha (TNF-
) is a multifunctional pro-inflammatory cytokine that is involved in the pathogenesis of a large number of autoimmune and inflammatory human diseases. The TNF- gene is located in the chromosomal region 6p21.1–21.3, next to the major histocompatibility complex. Increased TNF- expression in adipose tissue is associated with human obesity (Hotamisligil, 1999
). TNF- acts in a paracrine manner to interfere with insulin signalling by inhibiting autophosphorylation of the insulin receptor in fat and muscle cells (Hotamisligil et al., 1994a,b). It has been suggested to play an important role in insulin resistance and various components of the metabolic syndrome (Hotamisligil, 1999; Chan et al., 2002).
Pre-eclampsia is a common and complex disease with a familial nature and a genetic background (Lachmeijer et al., 2002). Impaired maternal immune response and different features of the metabolic syndrome, including insulin resistance, obesity, dyslipidemia and an increased risk of cardiovascular diseases are also characteristics of pre-eclampsia (Kilpatrick, 1996). In addition, previous studies have demonstrated elevated circulating concentrations of TNF- in patients with severe pre-eclampsia and intrauterine growth restriction (Kupferminc et al., 1994; Schiff et al., 1994; Kocyigit et al., 2004; Hayashi et al., 2005), and increased expression of the TNF- gene in patients with pre-eclampsia (Chen et al., 1996; Rinehart et al., 1999). TNF- has also been shown to affect early events in pregnancy, leading to failed trophoblast invasion and placentation (Bauer et al., 2004).
The promoter region of the TNF- gene has a single nucleotide polymorphism G-308A, which has been shown to be associated with elevated TNF- levels and a number of infectious and metabolic diseases (Abraham and Kroeger, 1999; Winchester et al., 2000; Pihlajamaki et al., 2003; Bayley et al., 2004; Rodriguez-Carreon et al., 2005). The G-308A polymorphism has also been found to be associated with the risk of pre-eclampsia in some studies (Chen et al., 1996), whereas others have shown no evidence for association (Dizon-Townson et al., 1998; Lachmeijer et al., 2001; Freeman et al., 2004). The G-308A allele of the TNF- gene has been reported to be associated with a 2-fold risk of type 2 diabetes, compared with the G-308G genotype (Kubaszek et al., 2003), and with an increased risk for the development of insulin resistance in obese subjects (Dalziel et al., 2002). Furthermore, the G-308A polymorphism has also been suggested to contribute to the risk of cardiovascular diseases in patients with type 2 diabetes (Vendrell et al., 2003). However, the association between the rare G-308A allele and the components of the metabolic syndrome has been questioned in some previous studies (Lee et al., 2000).
Another polymorphism, C-850T in the promoter region of the TNF- gene, has recently been connected with pre-eclampsia. We have previously reported that the C-850T allele may be protective against the development of pre-eclampsia (Heiskanen et al., 2002). In this study, we determined whether the haplotype of the two polymorphisms, C-850T and G-308A, in the promoter region of the TNF- gene, is associated with the risk of pre-eclampsia, using previously published and unpublished genotyping data from the same Finnish women.
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Materials and methods |
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Written approval for the study was obtained from the Ethics Committee of the Kuopio University Hospital. Informed consent was obtained from all patients and controls.
Information was collected retrospectively in connection with 133 pre-eclamptic pregnancies of primiparous women and 115 control women with no history of pre-eclampsia. They all delivered at the Kuopio University Hospital between January 1994 and December 1998. Pre-eclamptic patients were collected from the Birth Registry at Kuopio. They were called back and at that time asked to sign an informed consent. During the same time, blood samples were collected from the control women who had given birth in the same hospital after uncomplicated pregnancies and who had at least two normal pregnancies, including the current one. The controls had no clinical signs of the disorder. They originated from a regional population and were enrolled by random selection in this study.
Pre-eclampsia was defined as the development of hypertension and new-onset proteinuria (>300 mg of urinary protein in 24 h) in women with no proteinuria at baseline. Hypertension was defined as 
140 mmHg systolic and/or 90 mmHg systolic and diastolic blood pressure, when measured on two consecutive occasions at least 24 h apart (Report of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy, 2000). Women with previously diagnosed hypertension were excluded from the study.
The G-308A polymorphism of the TNF- gene was detected by the restriction fragment-length polymorphism (RFLP) method after PCR and digestion with the NcoI restriction enzyme, as previously described (Fernandez-Real et al., 1997).
Two-tailed pooled t-tests were used to analyse continuous variables. Statistical analyses for comparing single point allele and genotype frequencies were carried out using Pearson's 
2-test (two-sided asymptotic P values), with SPSS 9.0 software. Odds ratios, as the estimates of relative risk of disease, were calculated using 95% confidence intervals (CIs).
Two-locus haplotype frequencies of the TNF- promoter polymorphisms were estimated from genotype data with a maximum-likelihood method using an expectation-maximization algorithm under the assumption of Hardy–Weinberg equilibrium. Haplotype frequency comparisons between the pre-eclampsia and control groups, with absolute chromosome numbers, were carried out using an RxC program that employs the Metropolis algorithm. This program was used for analysis of contingency tables to obtain unbiased estimates of exact P values with standard errors in haplotype analyses. Hardy–Weinberg distributions of genotypes in the pre-eclampsia and control groups, as well as pairwise linkage disequilibrium (LD) analyses, were assessed using Arlequin ver. 2.000 software (Genetics and Biometry Lab, Department of Anthropology, University of Geneva, Switzerland). The level of statistical significance was defined as P<0.05. Sample size and power determinations were performed using nQuery Advisor Release 3.0 software (Statistical solutions, Saugus, MA). The frequency data was used to calculate the necessary sample sizes to achieve a power of 80% and an alpha of 0.05.
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Results |
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The clinical characteristics of the pre-eclamptic women and controls are shown in Table I. All women with pre-eclampsia were nulliparas, whereas the healthy control women had two or more deliveries. However, in the control subjects, the case records of their first pregnancies were used for comparison to exclude the confounding effect of parity on pregnancy outcome. On average, deliveries occurred 5 weeks earlier in the pre-eclamptic women than in the controls. There were no cases with HELLP (hemolysis, elevated liver enzymes and low platelet count) syndrome. When only the first-degree relatives of the index patients were taken into account, a positive family history was reported in 30 affected women, who had a total of six affected sisters and 28 affected mothers.
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The genotype and allele frequencies of the C-850T and G-308A polymorphisms in the promoter region of the TNF-
gene are presented in Table II. The genotype and allele distributions of the C-850T polymorphism differed significantly between the pre-eclampsia group and the control group (P=0.03 and P=0.03, respectively), as previously reported (Heiskanen et al., 2002). In the case of the G-308A polymorphism, there were no statistically significant differences in the genotype distribution (P=0.08) between the groups. According to the power calculations, we would have needed about 500 women in each group to achieve the power of 80% and an alpha of 0.05. The frequency of the A allele was 15% in the pre-eclampsia group and 9.1% among the controls (P=0.046), giving an odds ratio of 1.75 (95% CI: 1.01–3.13). The G-308A genotypes in both the study and control groups were found to be in Hardy–Weinberg equilibrium (P=0.062 and P=0.595, respectively).
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There is evidence that in complex diseases, statistical analysis based on haplotypes can provide additional power in detecting association over the single locus analysis (Martin et al., 2000
). We used the genotype data of the C-850T and G-308A polymorphisms in the pre-eclampsia and the control group to carry out a haplotype estimation analysis (Table III). Estimated overall pair of loci haplotype frequencies differed significantly between the groups (P=0.023±0.004). In the single haplotype association analysis, the haplotype C-A versus other pooled haplotypes was significantly over-represented in the pre-eclampsia group (P=0.041±0.003), whereas the haplotype T-G versus other pooled haplotypes was significantly less common in the pre-eclampsia group (P=0.035±0.003), compared with the controls.
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We also performed pairwise LD analysis to study the extent of LD between the C-850T and G-308A polymorphisms. No LD between the pairs of loci in the pre-eclampsia and control groups was found.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study, the overall haplotype of the two polymorphisms, C-850T and G-308A, in the promoter region of the TNF-
gene, was significantly associated with pre-eclampsia in Finnish women. The haplotype C-A was associated with an increased risk of pre-eclampsia, whereas the haplotype T-G was found to be protective. In addition, the less frequent A allele of the G-308A polymorphism was found to be associated with an increased risk of pre-eclampsia. The association between the C-850T polymorphism and pre-eclampsia has previously been published, indicating that the C-850T allele protects against the condition (Heiskanen et al., 2002). Thus, the present study gives further evidence that the TNF- gene plays a role in susceptibility to pre-eclampsia.
Besides its potent proinflammatory effects, TNF- has multiple metabolic effects that are mediated through impairment of insulin signalling in adipocytes (Hotamisligil et al., 1994a,b; Hotamisligil, 1999). Both TNF- gene expression and pre-eclampsia are associated with different features of the metabolic syndrome, including insulin resistance (Kaaja, 1998; Chan et al., 2002). Furthermore, overexpression of the TNF- gene is a feature of pre-eclampsia and intrauterine growth restriction, and TNF- serum concentrations have been shown to be often elevated in these conditions (Kupferminc et al., 1994; Schiff et al., 1994; Chen et al., 1996; Rinehart et al., 1999; Kocyigit et al., 2004; Rigo et al., 2004; Hayashi et al., 2005). However, TNF- has powerful local and tissue-specific paracrine effects which may be more important than its systemic exposure (Hotamisligil et al., 1995). Therefore, the serum concentrations of TNF- measured near term from pre-eclamptic women may be uninformative or even misleading with regard to its possible pathologic role which may be related to early pregnancy events under hypoxic conditions, including trophoblast invasion (Hayashi et al., 2005).
It is not known whether the C-850T polymorphism is biologically functional in the TNF- gene, whereas the less frequent G-308A allele of the TNF- gene is known to increase TNF- transcription and secretion under certain physiological conditions (Bouma et al., 1996; Wilson et al., 1997; Abraham and Kroeger, 1999). The G-308A allele has also been found to be associated with an increased risk of type 2 diabetes, dyslipidemia and coronary heart disease (Dalziel et al., 2002; Kubaszek et al., 2003; Vendrell et al., 2003), that are all related to pre-eclampsia in the long term. However, the results of previous studies on the effects of the G-308A polymorphism on the risk of pre-eclampsia have been ambiguous. Chen et al. (1996) found a significant association between the -308G allele and pre-eclampsia, whereas other investigators have failed to find such an association (Dizon-Townson et al., 1998; Lachmeijer et al., 2001; Freeman et al., 2004).
In summary, in this study, we demonstrated a haplotype association between pre-eclampsia and the two polymorphisms in the promoter region of the TNF- gene. The mechanisms behind this finding remain to be determined. They may be related to metabolic abnormalities increasing the risk of pre-eclampsia, or inflammatory effects caused by TNF-, or both. Further studies are needed to replicate our findings in other populations and to determine the exact molecular basis of this observation.
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Submitted on April 5, 2005; accepted on April 25, 2005.
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