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Keywords:

  • fetus;
  • genetic polymorphism;
  • mothers;
  • pre-eclampsia;
  • TNFR2

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References

Aim

The tumor necrosis factor receptor 2 (TNFR2) is expressed in placental tissue and it is involved in immune responses, inflammation, angiogenesis and blood pressure regulation; which makes it an attractive pre-eclampsia (PE) candidate gene. Furthermore, TNFR2 expression is altered in the first trimester in placentas of women who are destined to develop PE. Therefore, we examined the association between maternal and fetal genetic variants of TNFR2 and PE.

Material and Methods

Women with PE (n = 157) and their offspring with PE (n = 60) were compared to a control group of women (n = 97) and their offspring (n = 52) in the same Tunisian hospital-based population. We genotyped by polymerase chain reaction and restriction fragment length polymorphism the T/G polymorphism at position 676 in exon 6 (rs1061622) of the TNFR2 gene and examined its association with PE.

Results

The frequencies of TNFR2 (G/G) genotype and G allele were higher in the mothers with PE (n = 154) compared to the control group (15.3% vs 4.1% and 37% vs 26.3%, respectively); furthermore, the difference reached statistical significance (P = 0.002, odds ratio = 4.9; 95% confidence interval: 1.69–17.4 and P = 0.03, odds ratio = 1.69; 95% confidence interval: 1.03–2.8, respectively). In contrast, the fetal genotype and allele frequencies of this polymorphism had no effect on the risk of PE.

Conclusions

The exon 6 polymorphism in TNFR2 (rs1061622) or a gene at proximity is associated specifically with PE at least in the Tunisian population and could increase the risk for PE for mothers carrying the homozygote minor allele. Nevertheless, these results need to be confirmed in other populations.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References

Pre-eclampsia (PE) is a complex and serious disorder of human pregnancy defined by the presence of hypertension and proteinuria, which typically occurs after 20 weeks of gestation but improves after delivery.[1] In severe cases of PE, the only effective treatment is delivery, regardless of gestational age. Nevertheless, PE is a leading cause of maternal mortality as well as prenatal morbidity and mortality worldwide. This disorder affects at least 5–7% of all pregnancies in the world,[2] although the incidence is expected to be lower in pregnancies of parous women or in countries with lower rates of obesity. The cause is complex and both genetics and environmental factors influence the risk of developing this disease. Studies of the familial aggregation of PE indicate that the contribution of genetic factors could be greater than 31% of the variability in liability to PE.[3] First-degree female relatives of women who had PE have a fivefold higher risk and second-degree relatives have a twofold higher risk of developing PE during pregnancy compared to women without a family history of PE.[4] Paternal genetic factors, expressed through the fetus, may also contribute to an increased risk of PE.[5] The mechanisms of this disease proposed for the genesis of PE include poor placental vascular remodeling, abnormal placentation and thrombosis, endothelial cell dysfunction, oxidative stress, excessive intravascular inflammation, and the loss of endogenous protective regulators.[6] Numerous candidate genes have been evaluated for an association with PE, including those of the angiogenesis and inflammatory reaction.[7] The tumor necrosis factor (TNF) system is interesting because the vascular endothelial cells are among its principal physiological targets.[8] TNF-α exerts its effects by interacting with two receptors, which have distinct biological effects: the 55 kDa TNF receptor 1 (TNFR1) is believed to induce inflammation and apoptosis, while the 75-kDa TNF receptor 2 (TNFR2) serves to enhance TNFR1-induced cell death or to promote cell activation, proliferation or migration. TNFR1 is expressed ubiquitously, whereas TNFR2 expression is tightly regulated and found predominantly on endothelial, hematopoietic and neuronal cells as well as in partial expression. TNFR2 is mentioned in decidual immune cells;[9] furthermore, the cells can rapidly release soluble TNFR (sTNFR1 and sTNFR2) to give plasma-soluble sTNFR, which neutralizes TNF at high concentration.[10] sTNFR is expressed not only in the placenta but also in the amniotic fluid and urine[11, 12] of pregnant women, as well as in the plasma of women with a history of severe PE.[13] The TNFR2 gene carries several polymorphisms, including a functional one localized in exon 6 associated with an impaired NF-KB signaling and cell survival without affecting the TNF binding kinetics.[14] Being located in the fourth cysteine-rich domain of the receptor extracellular domain, this polymorphism might affect the binding site for TNFα-converting enzyme and the levels and function of sTNFR.[15] Moreover, this TNFR2 polymorphism has been genetically associated with an increased susceptibility of the development of chronic inflammatory disorders.[16] When studied together, all of the above observations suggest that the TNFR2 gene could be implicated in the pathogenesis of PE. To verify this theory, we conducted a case–control study in order to display an association between the polymorphism (676 G/T) in the TNFR2 gene and PE.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References

Study subjects

A prospective case–control study was conducted with unrelated cases of clinically-defined PE (157 maternal and 60 fetal) and controls (97 maternal and 52 fetal) selected from the same population living in the Middle Coast of Tunisia and recruited from the gynecological department of Monastir Hospital, Tunisia. A case was defined as a gravid woman with blood pressure 140/90 mm Hg or above (measured using mercury sphygmomanometers or electronic devices calibrated against a mercury standard), and proteinuria 0.3 g or above in 24 h, or a reading 2 or above on a dipstick in a random urine determination with no evidence of urinary tract infection, occurring after 20 weeks of gestation. At the time of the delivery, a verbal interview based on a structured questionnaire was conducted by trained personnel to ascertain maternal age, gestational age, parity, smoking status during pregnancy, family history of PE, personal history of PE and ethnic background.

A control was defined as a gravid woman without obstetrical complications or hypertensive problems, who was recruited from the same hospital that provided the cases. To improve the homogeneity of this phenotype under evaluation, women with a prior history of autoimmune, metabolic (including diabetes or gestational diabetes), renal, or cardiac (including chronic hypertension) diseases were excluded from this study. Also, only single pregnancies are considered in this analysis. This study was approved by the Hospital Ethics Committee and informed written consent was obtained from all participants.

DNA extraction

DNA was collected from the mothers via venous blood and their infants via umbilical cord blood. Venous blood from each subject was drawn into vacutainer tubes containing ethylenediaminetetraacetic acid and stored at 4°C. Genomic DNA was extracted from peripheral blood leukocytes by using the salting-out procedure.[17] Briefly explained, 5 mL of blood was mixed with Triton lysis buffer (0.32 M sucrose, 1% Triton X100, 5 mM MgCl2, 10 mM Tris-HCl pH 7.5). Leukocytes were spun down and washed with H2O. The pellet was incubated with proteinase K at 56°C and subsequently salted-out at 4°C using a saturated NaCl solution. Precipitated proteins were removed by centrifugation. The DNA in the supernatant fluid was precipitated with ethanol. The DNA pellet was dissolved in 300 μL H2O and stored at −20°C to be used as templates for polymerase chain reaction (PCR).

PCR and restriction fragment length polymorphism

The NlaIII polymorphism of TNFR2 genotype (rs1061622) was determined by PCR-restriction fragment length polymorphism assay. The sequences of primers used to amplify the TNFR2 genotype were 3′ forward primer (5′-ACTCTCCTATCCTGCCTGCT-3′) and 5′ reverse primer (5′-TTCTGGAGTTGGCTGCGTGT-3′). PCR was performed in a 25-μL volume containing 100 ng genomic DNA as template, 1X Taq polymerase buffer, 0.5 U of Taq DNA polymerase, 2 mM dNTPs, 1.5 mM MgCl2 and 0.75 μM of each primer. The PCR cycling conditions were 5 min at 95°C followed by 35 cycles of 1 min at 95°C, 1 min at 57°C and 1 min at 72°C, with a final step at 72°C for 10 min allowing a complete extension of all PCR fragments. A 10-μL aliquot of PCR product was subjected to digestion at 37°C for 16 h in a 15-μL reaction containing 1 U of NlaIII and 1.5-μL 10 × buffers. After digestion, the products were separated on a 3% agarose gel electrophoresis stained with ethidium bromide. The presence of an NlaIII site was indicated by the cleavage of the amplified product to yield two fragments of 133 and 109 bp. As a result, the wild homozygote (T/T) was represented by DNA bands of 133 and 109 bp and variant homozygote (G/G) by a DNA band of 242 bp, whereas the heterozygote (G/T) displayed a combination of both alleles (242, 133 and 109 bp).

Statistical analysis

The allele frequencies of TNFR2 were tested for the Hardy–Weinberg equilibrium for both patients and control groups using the χ2-test with one degree of freedom. The same test was used to evaluate for significant association between disease (PE against controls) and TNFR2 alleles or genotypes. The relative risk of PE associated with a particular genotype was estimated by the odds ratio (OR). The χ2-test (or Fisher's exact test when n < 5) was used to analyze the association of the TNFR2 polymorphism with the epidemiologic and clinical parameters. Statistical analyses were performed using spss 15. Differences between groups were tested for significance with the two-tailed test. A P-value of less than 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References

The demographic and clinical features of the women and neonates from pregnancies with PE are shown in Table 1. Among the cases, 111 (89.5%) met the criteria for mild PE (without maternal complications) and 13 (10.5%) were classified as severe PE. The single nucleotide polymorphism (SNP) studied was in Hardy–Weinberg equilibrium in all the controls and patient groups (mothers and children). In the maternal group, the genotype and allele frequencies of the TNFR2 polymorphism in the control and PE groups are shown in Table 2. Maternal cases specifically were more likely than controls to be carriers of the minor allele two copies for rs1061622 (15.3% vs 4.1%, P = 0.005). The frequency of G allele was also significantly higher in the patient group compared to the controls (37% vs 26.3%, P = 0.01). In contrast, there were no significant differences in the fetal genotype and allele frequencies of the TNFR2 T676G polymorphism between the cases and controls, showing that the offspring genotype of this SNP is not associated with PE (Table 3). In a statistical approximation, an association between genotype/allele frequencies and severity of the disease was not found. To test the association between PE onset and TNFR2 genotype variants, the group of PE patients was classified according to parity, age at onset, blood pressure, maternal and fetal complications, birthweight, hypertension and proteinuria. Again, no association between TNFR2 genotypes and clinical characteristics of PE was found (P-values > 0.05; Table 4).

Table 1. Demographic and clinical characteristics of 157 mothers with PE
Characteristicsn (%)
  1. Total number in some categories is less than 157 because of missing values. †Eclampsia; Hemolysis, Elevated Liver enzymes, Low Platelets syndrome; retroplacental hematoma. ‡Intrauterine growth restriction, intrauterine fetal death, neonatal death. PE, pre-eclampsia.

Maternal age of onset 
18–30 years54(42.5)
>30 years73(57.5)
Clinical subtypes 
Maternal complications 
Without111(89.5)
With13(10.5)
Fetal complication 
Without98(79)
With26(21)
Parity 
Primiparous46(36.2)
Multiparous81(63.8)
Birthweight 
<2000 g112(88.2)
>2000 g15(11.8)
Blood pressure 
<16/11101(79.5)
>16/1126(20.5)
Proteinuria 
<300 mg54(46)
>300 mg67(54)
Gestational age at delivery 
>36 weeks111(87.4)
<36 weeks16(12.6)
Personal history of PE 
With59(47.6)
Without65(52.4)
Family history of PE 
With75(60.5)
Without49(39.5)
Table 2. Maternal genotype and allele frequencies of TNFR2 (rs1061622) polymorphisms and risk of PE
 Controls (n = 97)Cases (n = 157)OR95%CIP-value
n (%)n (%)
  1. The χ2-test was used to determine whether significant differences(P-value) were observed when patient group was compared with control group. Significant at the levels: **P < 0.01; *P < 0.05. †The association remains significant under the recessive model(genotype TNFR2 G/G compared to genotype TNFR2 T/T + T/G) with OR = 4.17, P = 0.007, 95%CI(1.37–17.1). CI, confidence interval; OR, odds ratio; PE, pre-eclampsia; TNFR2, tumor necrosis factor receptor 2.

TNFR2 genotype     
T/T50(51.6)65(41.4)1  
T/G43(44.3)68(43.3)1.210.71 < OR < 2.070.55
G/G4(4.1)24(15.3)4.51.44 < OR < 19.30.005**
TNFR2 allele frequencies     
TNFR2-T(73.7)(63)1  
TNFR2-G(26.3)(37)1.641.11 < OR < 2.440.01*
Table 3. Fetal genotype and allele frequencies of TNFR2 (rs1061622) polymorphisms and risk of PE
 Controls (n = 52)Cases (n = 60)OR95%CIP-value
n (%)n (%)
  1. The χ2-test was used to determine whether significant differences(P-value) were observed when patient group was compared with control group. †The association remains not significant under the recessive model(genotype TNFR2 G/G compared to genotype TNFR2 T/T + T/G) with OR = 1.16, 95%CI(0.2–5.4) and P = 0.84. CI, confidence interval; OR, odds ratio; PE, pre-eclampsia; TNFR2, tumor necrosis factor receptor 2.

TNFR2 genotype     
T/T27(51.9)33(55)1  
T/G21(40.4)23(38.3)1.110.5 < OR < 2.40.78
G/G4(7.7)4(6.6)1.210.25 < OR < 5.80.79
TNFR2 allele frequencies     
TNFR2-T(72.1)(74)1  
TNFR2-G(27.9)(26)1.110.6 < OR < 20.73
Table 4. Association between the maternal genotype frequencies of tumor necrosis factor receptor 2 (676T/G) polymorphisms and clinical parameters of pre-eclampsia
 Genotypes n (%)P-value
TTTGGG
  1. Total number in some categories is less than 154 because of missing values. The χ2-test was used to determine whether significant differences(P-value) were observed when patient group was subdivided according the clinical parameters of PE. †Eclampsia; Hemolysis, Elevated Liver enzymes, Low Platelets syndrome; retroplacental hematoma. ‡Intrauterine growth restriction, intrauterine fetal death, neonatal death.

Maternal age of onset    
18–30 years(n = 55)23(0.42)24(0.43)8(0.14) 
>30 years(n = 74)31(0.42)34(0.46)9(0.12)0.91
Maternal complications    
Without(n = 110)44(0.38)50(0.42)21(0.14) 
With(n = 17)8(0.53)8(0.38)1(0.06)0.6
Fetal complication    
Without(n = 102)41(0.4)50(0.49)11(0.11) 
With(n = 26)11(0.44)8(0.32)7(0.24)0.14
Parity    
Primiparous(n = 48)20(0.42)16(0.39)10(0.19) 
Multiparous(n = 81)34(0.42)39(0.48)8(0.10)0.31
Birthweight    
>2000 g(n = 112)44(0.39)52(0.46)16(0.14) 
<2000 g(n = 17)10(0.59)6(0.35)1(0.06)0.28
Blood pressure    
<16/11(n = 99)41(0.41)45(0.45)13(0.13) 
>16/11(n = 30)13(0.43)13(0.43)4(0.13)0.97
Proteinuria    
<300 mg(n = 58)25(0.43)29(0.5)4(0.07) 
>300 mg(n = 69)27(0.39)29(0.42)13(0.19)0.14

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References

To the best of our knowledge, this is the first study looking for an association between TNFR2 genetic polymorphism and PE. Interestingly, we found a significant correlation between the maternal genetic polymorphisms of TNFR2 rs1061622 (which is in strong linkage disequilibrium with the CA-repeat polymorphism) and PE. On the other hand, there was no link between the same fetal genetic variant and the disease. These results suggest that this TNFR2 variant allele or a polymorphism in proximity could be implicated in PE development. The cause and pathogenesis of PE are not completely understood, despite extensive research. This disorder is thought to be multifactorial in origin, with multiple genes, environmental and social factors contributing to the disease. One proposed mechanism is thought to be the consequence of impaired trophoblastic invasion of the maternal spiral arteries.[18] This leads to placental hypoxia and the release of inflammatory factors that cause endothelial cell dysfunction.[19] There is an increasing body of evidence that an exaggerated maternal systemic inflammatory response to pregnancy plays a central role in the pathogenesis of the disease.[20] The excessive production of proinflammatory cytokines, chemokines and adhesion molecules may trigger a generalized endothelial dysfunction characteristic of the maternal syndrome of PE. Among the inflammatory molecules, the TNF pleiotropic inflammatory cytokine is assumed to play significant physiological and pathological roles in the placenta.[7] It is generally accepted that TNF utilizes TNFR1 and TNFR2 to trigger distinct signal transduction pathways and to exert diverse biological functions in a context-dependant manner, including apoptosis, proliferation and differentiation. Under certain circumstances, TNFR2 may contribute to TNFR1 responses, particularly at low concentrations of TNF,[21] consistent with the notion of ‘ligand passing’, in which TNFR2 captures TNF and passes it to TNFR1. Cooperation between the receptors may also be explained by the ligand-induced formation of TNF receptor heterocomplexes leading to cell death.[22] It has been shown that in T cells, TNF-TNFR2 promotes proliferation.[23] In oligodendrocytes, TNFR2 is critical in TNF-induced proliferation of progenitors and remyelination.[24] In endothelial cells, TNFR2 induce in vitro, via Etk-VEGFR2 cross-talk, migration and tube formation, implying that this receptor may play a critical role in inflammatory angiogenesis, such as the ones occurring with ischemia, atherosclerosis or PE.[25] In normal pregnancy, the TNFR2 protein is expressed in first-trimester cytotrophoblasts and syncytium with decreasing levels in these cell types toward the end of pregnancy.[26] In the PE, the implication of TNFR2 is supported by studies showing that elevated levels of sTNFR2 are prior to overt PE[27] and at the diagnostic stage.[13] Moreover, elevated serum levels of TNF,[28] as well as increased mRNA/protein expression of TNF/TNFR were noticed in the leukocytes[29] and placenta[30] of PE women. Functional analyses from mice genetically deficient of TNFR demonstrate that TNFR1 and TNFR2 play differential roles in ischemia-mediated arteriogenesis and angiogenesis. The TNFR signaling analysis indicated that TNFR1 induce decreased arteriogenesis, angiogenesis, and associated endothelial cell proliferation, neovascularization, and vessel maturation, whereas TNFR2 induce an increase.[31-34] The TNFR2 gene is located on chromosome 1p36.2 and is organized in 10 exons and nine introns.[35] In addition to non-coding SNP in exons 4, 9, and 10, a further SNP (T/G) was described in exon 6 at nucleotide 676 of the TNFR2 mRNA resulting in an amino acid exchange in the fourth extracellular cysteine-rich domain (CRD4) from methionine (TNFR2 196MET) to arginine (TNFR2 196ARG) at position 196. Exon 6 encodes a small portion of the transmembrane region and contains the position of the proteolytic cleavage site that produces the soluble form of TNFR2. Receptor shedding provides a mechanism for downregulating a cell surface receptor and a means of releasing a biologically active, soluble receptor, which may act as a receptor-antagonist by capturing free-circulating ligand.[36] Investigations of the functional impact of a single nucleotide polymorphism in exon 6 of the TNFR2 gene demonstrated that the TNFR2 196ARG risk variant has a significantly lower capability to induce direct NF-kB signaled via TNFR2 in human epithelial cells. The diminished capability of the mutated TNFR2 196ARG to induce NF-kB activation is paralleled by a diminished induction of NF-kB-dependent target genes conveying either anti-apoptotic or pro-inflammatory functions, such as cIAP1, cIAP2, TRAF1, IL-6, and IL-8.[37] Moreover, T676G polymorphism in TNFR2 is associated with levels of sTNFR released from peripheral blood T cells, and with circulating levels of sTNFR in patients with rheumatoid arthritis; however physical binding parameters appear to be not influenced by the substitution.[14, 15] The functional impact of this SNP remains to be demonstrated in endothelial cells; however, if we hypothesized that the effect of this SNP, described in epithelial cells,[37] could be similar in endothelial and bone marrow-derived cells, the TNFR2 (196R) variant could reduce signaling receptors leading to reduced survival, migration and tube formation and contribute to arteriogenesis and angiogenesis in local placenta. Subsequently, this polymorphism could lead to a poor placentation and to the endothelial dysfunction described in PE. Thus, this polymorphism could contribute to explaining the risk of PE, at least for Tunisian women. Future studies among different ethnic populations are needed to determine whether our results can be extended to other ethnic groups.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References

No author has any potential conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References