Tibor Várkonyi

Microarray profiling reveals that placental transcriptomes of early-onset HELLP syndrome and preeclampsia are similar

BACKGROUND The involvement of the placenta in the pathogenesis of preeclampsia and HELLP syndrome is well established, and placental lesions are also similar in these two syndromes. Here we aimed to examine the placental transcriptome and to identify candidate biomarkers in early-onset preeclampsia and HELLP syndrome. METHODS Placental specimens were obtained at C-sections from women with early-onset preeclampsia and HELLP syndrome, and from controls who delivered preterm or at term. After histopathological examination, fresh-frozen placental specimens were used for microarray profiling and validation by qRT-PCR. Differential expression was analysed using log-linear models while adjusting for gestational age. Gene ontology and pathway analyses were used to interpret gene expression changes. Tissue microarrays were constructed from paraffin-embedded placental specimens and immunostained. RESULTS Placental gene expression was gestational age-dependent among preterm and term controls. Out of the 350 differentially expressed genes in preeclampsia and 554 genes in HELLP syndrome, 224 genes (including LEP, CGB, LHB, INHA, SIGLEC6, PAPPA2, TREM1, and FLT1) changed in the same direction (elevated or reduced) in both syndromes. Many of these encode proteins that have been implicated as biomarkers for preeclampsia. Enrichment analyses revealed similar biological processes, cellular compartments and biological pathways enriched in early-onset preeclampsia and HELLP syndrome; however, some processes and pathways (e.g., cytokine-cytokine receptor interaction) were over-represented only in HELLP syndrome. CONCLUSION High-throughput transcriptional and tissue microarray expression profiling revealed that placental transcriptomes of early-onset preeclampsia and HELLP syndrome largely overlap, underlying a potential common cause and pathophysiologic processes in these syndromes. However, gene expression changes may also suggest a more severe placental pathology and pronounced inflammatory response in HELLP syndrome than in preeclampsia.

Association between tumor necrosis factor (TNF)-α G-308A gene polymorphism and preeclampsia complicated by severe fetal growth restriction

BACKGROUND Preeclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome are multifactorial disorders with genetic and environmental components. Given that the tumor necrosis factor (TNF)-alpha G-308A single nucleotide polymorphism (SNP) affects TNF-alpha gene transcription and that preeclampsia and HELLP syndrome are characterized by a shift towards a Th1-type maternal immune response with increased TNF-alpha production, the aim of the current study was to investigate whether this SNP is associated with preeclampsia and HELLP syndrome in a Caucasian population from Hungary. Additionally, we aimed to examine whether TNF-alpha G-308A polymorphism can influence the risk for fetal growth restriction in preeclamptic patients, which issue none of the earlier studies dealt with. METHODS In a case-control study, we analyzed blood samples from 140 preeclamptic patients, 69 patients with HELLP syndrome and 144 normotensive, healthy pregnant women using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. We performed also a meta-analysis with our results and those of 8 previously published studies. RESULTS There were no significant differences in the genotype and allele frequencies of the TNF-alpha G-308A polymorphism between preeclamptic patients and normotensive, healthy pregnant women. However, the mutant (TNF2 or A) allele occurred significantly more frequently in preeclamptic patients with IUGR than in those without IUGR (18.5% versus 7.1%, p=0.003). In addition, the frequency of the mutant allele carriers was significantly higher among preeclamptic patients with IUGR compared to those without IUGR (30.6% versus 12.8%, p=0.010). The mutant allele carriers were found to have an increased risk of severe IUGR-complicated preeclampsia, which was independent of maternal age, prepregnancy BMI and primiparity (odds ratio (OR): 2.89, 95% confidence interval (CI): 1.16-7.22, p=0.023; adjusted OR: 2.78, 95% CI: 1.04-7.45, p=0.042). Nevertheless, no significant differences were detected in the genotype and allele frequencies of the TNF-alpha G-308A polymorphism between patients with HELLP syndrome and control subjects. In the meta-analysis, no association was observed between this SNP and preeclampsia (summary OR: 0.956, 95% CI: 0.693-1.319). CONCLUSIONS Although the meta-analysis demonstrated a lack of an overall association between TNF-alpha G-308A polymorphism and preeclampsia, our results suggest a role of this SNP in the risk of severe IUGR-complicated preeclampsia. However, further studies are required with a larger sample size to confirm our findings.

Vascular endothelial growth factor (VEGF) polymorphisms in HELLP syndrome patients determined by quantitative real-time PCR and melting curve analyses

BACKGROUND The vascular endothelial growth factor (VEGF) has a critical role in vasculogenesis and vascular permeability in several diseases including preeclampsia. There are at least 30 single nucleotide polymorphic (SNP) places on this gene. VEGF G+405C, C-2578A and C-460T SNPs are known to be related to VEGF production. VEGF polymorphisms were studied in preeclampsia, but not in HELLP syndrome. Therefore, we decided to determine the allele and genotype frequencies of VEGF G+405C, C-460T and C-2578A SNPs in healthy pregnant women and HELLP syndrome patients. METHODS The authors introduced a quantitative real-time PCR method for the determination of the three VEGF SNPs. Blood samples were collected from 71 HELLP syndrome patients and 93 healthy controls. DNA was isolated by using silica adsorption method. The SNPs were determined by quantitative real-time PCR and melting curve analysis using LightCycler. RESULTS There were significant differences in the allele and genotype frequencies of VEGF C-460T SNP between the two study groups. The T allele was present in 71.1% in the HELLP group, while in 53.8% in the controls (p=0.0014). The TT genotype occurred significantly more frequently in the HELLP group than in the control group (45.1% vs. 21.5%; p (for genotype frequencies)=0.0011). The TT genotype carriers had an increased risk of HELLP syndrome, which was independent of maternal age and primiparity (adjusted odds ratio (OR)=3.03, 95% confidence interval (CI)=1.51-6.08; p=0.002). Although the VEGF G+405C allele and genotype distributions did not differ significantly between the two groups, the CC genotype carriers were also found to have an increased risk for HELLP syndrome after adjustment for maternal age and primiparity (adjusted OR=3.67, 95% CI=1.05-12.75; p=0.041). The VEGF C-2578A SNP was not associated with HELLP syndrome. CONCLUSIONS The quantitative real-time PCR combined with melting curve analyses is a fast and reliable method for the determination of VEGF SNPs. We found that the VEGF -460TT and +405CC genotype carriers have an increased risk of HELLP syndrome. As these two SNPs were previously observed to be related to production of the VEGF protein, we suppose that these VEGF polymorphisms -- interacting with other genetic and environmental factors - could play a role in the development of HELLP syndrome.

Leptin gene (TTTC)n microsatellite polymorphism in pre-eclampsia and HELLP syndrome

Abstract Background: Leptin plays an important role in energy homeostasis. There is polymorphism on the leptin (LEP) gene. Our aim was to compare the tetranucleotide repeat (TTTC)n polymorphism in the 3′-flanking region in the LEP gene on DNA samples from patients with pre-eclampsia (PE), hemolysis, elevated liver enzymes, and low platelet (HELLP) syndrome and healthy pregnant controls. Methods: Blood samples were collected from healthy pregnant women (n=88), patients with PE (n=79) and HELLP (n=77) syndrome. Fluorescent PCR and DNA fragment analysis was performed from the isolated DNA for the detection of (TTTC) repeats. The electrophoretograms were evaluated and patients were assigned to two groups; class I low (<190 bp) or class II high (≥190 bp) PCR fragments. Results: We observed similar distributions of the class I and class II (TTTC) alleles in the groups studied (class I allele: healthy pregnant 58.5%; severe pre-eclamptic 58.3%; HELLP syndrome 52.6%). We detected a higher frequency of the II/II genotype in HELLP syndrome patients (32.4%) compared to healthy controls (22.7%). However, the difference was not statistically significant. Conclusions: In an ethnically homogenous population, the LEP gene (TTTC) microsatellite polymorphism in the 3′-flanking region does not show a significant difference in the allele and genotype distribution in healthy pregnant, pre-eclamptic and HELLP syndrome patients. Furthermore, we recommend a new classification of the class I and class II alleles based on the distribution of the (TTTC) microsatellites. Clin Chem Lab Med 2009;47:1033–7.

Early-Onset Preeclampsia and HELLP Syndrome: An Overview

Preeclampsia, one of the “great obstetrical syndromes,” affects ~3–5% of pregnancies and is a major cause of maternal and perinatal morbidity and mortality. Preeclampsia is diagnosed after 20 weeks of gestation and is characterized by new-onset hypertension and proteinuria in previously normotensive women, which may deteriorate into maternal multiorgan damage affecting the kidneys, liver and central nervous system. This multisystem disorder is unique to human pregnancy and is the clinical manifestation of heterogeneous pathological processes. The placenta plays a key role in the underlying mechanisms leading to the development of preeclampsia as the only definitive treatment today remains delivery. Early-onset preeclampsia often has severe maternal and foetal consequences including intrauterine growth restriction, preterm delivery, low or very low birth weight, increased perinatal morbidity and mortality and a high incidence of the life-threatening HELLP syndrome, while the clinical presentation of late-onset preeclampsia is frequently mild, resulting mainly in maternal consequences. Of further importance, pregnant women with severe forms of preeclampsia and their growth-restricted foetuses are at an increased risk for developing cardiovascular disease later in life. Here we review the literature on the epidemiology, risk factors, pathophysiology, maternal and perinatal outcomes, diagnosis and management of early-onset preeclampsia and HELLP syndrome and summarize how these severe pregnancy complications are related to foetal growth and health.