Molecular genetic studies in pregnancies affected by preeclampsia and intrauterine growth restriction

Preeclampsia and fetal growth restriction (FGR) are common and costly obstetric complications. Both conditions are associated with immediate and remote mortality and morbidity for the mother and the offspring. Impaired placentation and aberrant maternal systemic responses are implicated as pathophysiological mechanisms in preeclampsia and FGR. Both preeclampsia and FGR are known to have a clear genetic basis. This study has investigated the roles of several candidate genes including those previously associated with diabetes (TCF7L2, FTO, PPAR-g, CDKN2B-AS1 and KCNJ11), and epidermal growth factor (EGF). Functional consequences of variants within the EGF gene were also investigated. A bidirectional association between type 2 diabetes (T2D) and preeclampsia is consistently reported, whereby each condition is associated with an increased risk of the other. Furthermore, fetal growth restriction, which complicates 30% of preeclamptic pregnancies, predisposes the offspring to an increased risk of type 2 diabetes and coronary artery disease (CAD) later in life. 11 single nucleotide polymorphisms (SNPs) reproducibly associated with T2D in the TCF7L2, FTO, PPAR-y, CDKN2B-AS1 and KCNJ11 genes were investigated as susceptibility loci for preeclampsia and fetal growth restriction in a maternal case control study. The study group consisted of 448 white western European women with preeclampsia, 673 controls with no evidence of preeclampsia, 243 women with pregnancies complicated by FGR, and 570 controls with no evidence of growth restriction. A maternal haplotype on the T2D region of the CDKN2BAS1 gene on chromosome 9p21 was found to be a risk variant for fetal growth restriction (P=O.005). The other 9 investigated SNPs in TCF7L2, FTO, PPAR-y, and KCNJ11 showed no association with growth restricted pregnancies. None of the SNPs investigated showed an association with preeclampsia. These findings suggest that some maternal diabetogenic risk variants are associated with an altered risk of FGR pregnancy but not preeclampsia. The results require replication in a larger sample and fetal-maternal gene interactions merit investigation. Epidermal growth factor (EGF) is described as a major regulator of the placentation process. It also helps to maintain an adequate blood supply to the growing fetus through its effects on umbilical vessel tone. Investigating the role of two genetic variants of the EGF gene in susceptibility to preeclampsia and FGR showed that the maternal variants, rs4444903 in the 5'UTR and rs2237051 in exon 14 of the EGF gene has no effect on the risk of preeclampsia or FGR pregnancy. The G allele of the SNP rs4444903 was associated with higher systolic blood pressure measures in the control group. The G allele of the rs4444903 and the A allele of rs2237051 have been associated with increased risk for FGR and lower birth weight in a previous study from our laboratory. This led to investigations to characterize the functional consequences of the two SNPs in the EGF gene on transcription, translation and ribonucleic acid (RNA) splicing using a variety of methods. These experiments have shown that the G allele of rs4444903 was transcriptionally more active than the A allele in hepatocellular carcinoma (HepG2) and more active than EGFP on its own in choriocarcinoma (Jeg-3) cell lines using a luciferase reporter gene assay. There was no effect of this variant on translational efficiency in the cell lines investigated using reporter gene assays, or in a cell free environment using an in vitro translation assay. DNA-protein interaction was investigated using nuclear extract from HepG2 cells to further define the mechanism by which the G allele exerts its higher transcriptional activity. Initial experiments suggest that the Sp1 transcription factor interacts with and represses the A allele of the rs4444903 SNP. The study also demonstrated no evidence of higher activity of the G allele on EGF expression in vivo using term placental tissues. It was expected that higher EGF expression as a function of genotype at rs4444903 SNP may lead to down regulation of the EGFR in the placenta, which was not confirmed in this study. SNP rs2237051 in exon 14 of the EGF gene is in strong linkage disequilibrium with rs4444903, and disrupts a predicted exon splicing enhancer region. This polymorphism was investigated using a minigene assay, but there was no evidence that it affected splicing of exon 14. Taken together, these findings provide no evidence that EGF genetic variants alter the risk of preeclampsia or FGR though functioning.