Gijs B. Afink

Molecular genetics of preeclampsia and HELLP syndrome - A review

Preeclampsia is characterised by new onset hypertension and proteinuria and is a major obstetrical problem for both mother and foetus. Haemolysis elevated liver enzymes and low platelets (HELLP) syndrome is an obstetrical emergency and most cases occur in the presence of preeclampsia. Preeclampsia and HELLP are complicated syndromes with a wide variety in severity of clinical symptoms and gestational age at onset. The pathophysiology depends not only on periconceptional conditions and the foetal and placental genotype, but also on the capability of the maternal system to deal with pregnancy. Genetically, preeclampsia is a complex disorder and despite numerous efforts no clear mode of inheritance has been established. A minor fraction of HELLP cases is caused by foetal homozygous LCHAD deficiency, but for most cases the genetic background has not been elucidated yet. At least 178 genes have been described in relation to preeclampsia or HELLP syndrome. Confined placental mosaicism (CPM) is documented to cause early onset preeclampsia in some cases; the overall contribution of CPM to the occurrence of preeclampsia has not been adequately investigated yet. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.

Molecular Characterization of Iodotyrosine Dehalogenase Deficiency in Patients with Hypothyroidism

CONTEXT The recent cloning of the human iodotyrosine deiodinase (IYD) gene enables the investigation of iodotyrosine dehalogenase deficiency, a form a primary hypothyroidism resulting from iodine wasting, at the molecular level. OBJECTIVE In the current study, we identify the genetic basis of dehalogenase deficiency in a consanguineous family. RESULTS Using HPLC tandem mass spectrometry, we developed a rapid, selective, and sensitive assay to detect 3-monoiodo-l-tyrosine and 3,5-diodo-l-tyrosine in urine and cell culture medium. Two subjects from a presumed dehalogenase-deficient family showed elevated urinary 3-monoiodo-l-tyrosine and 3,5-diodo-l-tyrosine levels compared with 57 normal subjects without thyroid disease. Subsequent analysis of IYD revealed a homozygous missense mutation in exon 4 (c.658G>A p.Ala220Thr) that co-segregates with the clinical phenotype in the family. Functional characterization of the mutant iodotyrosine dehalogenase protein showed that the mutation completely abolishes dehalogenase enzymatic activity. One of the heterozygous carriers for the inactivating mutation recently presented with overt hypothyroidism indicating dominant inheritance with incomplete penetration. Screening of 100 control alleles identified one allele positive for this mutation, suggesting that the c.658G>A nucleotide substitution might be a functional single nucleotide polymorphism. CONCLUSIONS This study describes a functional mutation within IYD, demonstrating the molecular basis of the iodine wasting form of congenital hypothyroidism. This familial genetic defect shows a dominant pattern of inheritance with incomplete penetration.

Differentially expressed genes in the pre-eclamptic placenta: a systematic review and meta-analysis.

Objective To systematically review the literature on human gene expression data of placental tissue in pre-eclampsia and to characterize a meta-signature of differentially expressed genes in order to identify novel putative diagnostic markers. Data Sources Medline through 11 February 2011 using MeSH terms and keywords related to placenta, gene expression and gene expression arrays; GEO database using the term “placent*”; and reference lists of eligible primary studies, without constraints. Methods From 1068 studies retrieved from the search, we included original publications that had performed gene expression array analyses of placental tissue in the third trimester and that reported on differentially expressed genes in pre-eclampsia versus normotensive controls. Two reviewers independently identified eligible studies, extracted descriptive and gene expression data and assessed study quality. Using a vote-counting method based on a comparative meta-profiling algorithm, we determined a meta-signature that characterizes the significant intersection of differentially expressed genes from the collection of independent gene signatures. Results We identified 33 eligible gene expression array studies of placental tissue in the 3rd trimester comprising 30 datasets on mRNA expression and 4 datasets on microRNA expression. The pre-eclamptic placental meta-signature consisted of 40 annotated gene transcripts and 17 microRNAs. At least half of the mRNA transcripts encode a protein that is secreted from the cell and could potentially serve as a biomarker. Conclusions In addition to well-known and validated genes, we identified 14 transcripts not reported previously in relation to pre-eclampsia of which the majority is also expressed in the 1st trimester placenta, and three encode a secreted protein.

Expression of Placental FLT1 Transcript Variants Relates to Both Gestational Hypertensive Disease and Fetal Growth

The recent discovery of additional alternative spliced FLT1 transcripts encoding novel soluble (s)FLT1 protein isoforms complicates both the predictive value and functional implications of sFLT1 in preeclampsia. We investigated FLT1 expression levels and splicing patterns in placentas of normotensive and preeclamptic women, and established the tissue specificity of all FLT1 transcript variants. mRNA levels of sFLT1 splice variants were determined by real-time polymerase chain reaction in 21 normal human tissues and placental biopsies from 91 normotensive and 55 preeclamptic women. Cellular localization of placental FLT1 expression was established by RNA in situ hybridization. Of all tissues investigated, placenta has by far the highest FLT1 mRNA expression level, mainly localized in the syncytiotrophoblast layer. More than 80% of placental transcripts correspond to sFLT1_v2. Compared with normotensive placenta, preeclamptic placenta has ≈3-fold higher expression of all FLT1 transcript variants (P<0.001), with a slight shift in favor of sFLT1_v1. Although to a lesser degree, transcript levels are also increased in placenta from normotensive women that deliver a small for gestational age neonate. We conclude that sFLT isoform–specific assays could potentially improve the accuracy of current sFLT1 assays for the prediction of preeclampsia. However, placental FLT1 transcript levels are increased not only in preeclampsia but also in normotensive pregnancy with a small for gestational age fetus. This may indicate a common pathway involved in the development of both conditions but complicates the use of circulating sFLT1 protein levels for the prediction or diagnosis of preeclampsia alone.

ELABELA deficiency promotes preeclampsia and cardiovascular malformations in mice

Modeling a pregnancy disorder Preeclampsia, a dangerous pregnancy disorder marked by high blood pressure, can lead to premature birth and be life-threatening to the mother and baby. Research leading to effective treatments has been hampered by a lack of informative animal models. Ho et al. identified ELABELA as a hormone produced by the placenta whose levels are lower in preeclampsia (see the Perspective by Wirka and Quertermous). ELABELA-deficient pregnant mice showed clinical signs of preeclampsia, including high blood pressure and elevated urine protein. A proportion of embryos lacking ELABELA displayed defective heart development, and full-term pups had low birth weights. Science, this issue p. 707; see also p. 643 ELABELA is a placental hormone that functions in preeclampsia and heart development during embryogenesis. Preeclampsia (PE) is a gestational hypertensive syndrome affecting between 5 and 8% of all pregnancies. Although PE is the leading cause of fetal and maternal morbidity and mortality, its molecular etiology is still unclear. Here, we show that ELABELA (ELA), an endogenous ligand of the apelin receptor (APLNR, or APJ), is a circulating hormone secreted by the placenta. Elabela but not Apelin knockout pregnant mice exhibit PE-like symptoms, including proteinuria and elevated blood pressure due to defective placental angiogenesis. In mice, infusion of exogenous ELA normalizes hypertension, proteinuria, and birth weight. ELA, which is abundant in human placentas, increases the invasiveness of trophoblast-like cells, suggesting that it enhances placental development to prevent PE. The ELA-APLNR signaling axis may offer a new paradigm for the treatment of common pregnancy-related complications, including PE.

Transglutaminase 2 is secreted from smooth muscle cells by transamidation-dependent microparticle formation

Transglutaminase 2 (TG2) is a pleiotropic enzyme involved in both intra- and extracellular processes. In the extracellular matrix, TG2 stabilizes the matrix by both covalent cross-linking and disulfide isomerase activity. These functions become especially apparent during matrix remodeling as seen in wound healing, tumor development and vascular remodeling. However, TG2 lacks the signal sequence for a classical secretory mechanism, and the cellular mechanism of TG2 secretion is currently unknown. We developed a green fluorescent TG2 fusion protein to study the hypothesis that TG2 is secreted via microparticles. Characterization of TG2/eGFP, using HEK/293T cells with a low endogenous TG2 expression, showed that cross-linking activity and fibronectin binding were unaffected. Transfection of TG2/eGFP into smooth muscle cells resulted in the formation of microparticles (MPs) enriched in TG2, as detected both by immunofluorescent microscopy and flow cytometry. The fraction of TG2-positive MPs was significantly lower for cross-linking-deficient mutants of TG2, implicating a functional role for TG2 in the formation of MPs. In conclusion, the current data suggest that TG2 is secreted from the cell via microparticles through a process regulated by TG2 cross-linking.

Brain abnormalities and glioma-like lesions in mice overexpressing the long isoform of PDGF-A in astrocytic cells.

Background Deregulation of platelet-derived growth factor (PDGF) signaling is a hallmark of malignant glioma. Two alternatively spliced PDGF-A mRNAs have been described, corresponding to a long (L) and a short (S) isoform of PDGF-A. In contrast to PDGF-A(S), the PDGF-A(L) isoform has a lysine and arginine rich carboxy-terminal extension that acts as an extracellular matrix retention motif. However, the exact role of PDGF-A(L) and how it functionally differs from the shorter isoform is not well understood. Methodology/Principal Findings We overexpressed PDGF-A(L) as a transgene under control of the glial fibrillary acidic protein (GFAP) promoter in the mouse brain. This directs expression of the transgene to astrocytic cells and GFAP expressing neural stem cells throughout the developing and adult central nervous system. Transgenic mice exhibited a phenotype with enlarged skull at approximately 6-16 weeks of age and they died between 1.5 months and 2 years of age. We detected an increased number of undifferentiated cells in all areas of transgene expression, such as in the subependymal zone around the lateral ventricle and in the cerebellar medulla. The cells stained positive for Pdgfr-α, Olig2 and NG2 but this population did only partially overlap with cells positive for Gfap and the transgene reporter. Interestingly, a few mice presented with overt neoplastic glioma-like lesions composed of both Olig2 and Gfap positive cell populations and with microvascular proliferation, in a wild-type p53 background. Conclusions Our findings show that PDGF-A(L) can induce accumulation of immature cells in the mouse brain. The strong expression of NG2, Pdgfr-α and Olig2 in PDGF-A(L) brains suggests that a fraction of these cells are oligodendrocyte progenitors. In addition, accumulation of fluid in the subarachnoid space and skull enlargement indicate that an increased intracranial pressure contributed to the observed lethality.

SFlt-1 elevates blood pressure by augmenting endothelin-1-mediated vasoconstriction in mice.

Objective Scavenging of vascular endothelial growth factor (VEGF) elevates blood pressure (BP) in patients receiving anti-angiogenic therapy. Similarly, inhibition of circulation VEGF by its soluble receptor fms-like tyrosine kinase-1 (sFlt-1) underlies BP elevation in pre-eclampsia. Both phenotypes are characterized by augmented production of endothelin-1 (ET-1), suggesting a role for ET-1 in anti-angiogenic hypertension. We aimed to assess the effect of VEGF inhibition on ET-1-induced contractility and downstream ET-1 signaling. Approach and Results Male C57BL/6N mice were treated with either sFlt-1 or vehicle and BP was assessed via tail-cuff. Mean arterial pressure of sFlt-1-treated mice markedly increased compared to vehicle-treated controls (N = 11–12, p<0.05). After sacrifice, carotid and mesenteric arteries were isolated for isometric tension measurements. ET-1-induced contractions were similar in mesenteric arteries of vehicle and sFlt-1-treated mice, but augmented in carotid segments of sFlt-1-treated mice compared to controls (N = 9–10, p<0.05). The increased contraction in carotid segments could be completely abrogated by the cyclooxygenase (COX) inhibitor indomethacin (N = 9–10, p<0.05), indicating heightened prostaglandin-mediated vasoconstriction. This was associated with a shift towards procontractile ETB signaling in sFlt-1-treated mice, possibly explaining the increased ET-1-induced prostaglandin-mediated vasoconstriction. In line with the ex vivo findings, sFlt-1-induced BP elevation could be prevented in vivo by oral treatment with either a high-dose of the COX inhibitor aspirin (N = 7) or with picotamide (N = 9), a dual thromboxane A2 synthase inhibitor and receptor antagonist. Conclusions VEGF inhibition augments the pressor response to ET-1. The cyclooxygenase-thromboxane signaling route downstream of ET-1 might be a possible target to prevent BP elevation during VEGF inhibition.

Resistance Artery Creatine Kinase mRNA and Blood Pressure in Humans

Hypertension remains the main risk factor for cardiovascular death. Environmental and biological factors are known to contribute to the condition, and circulating creatine kinase was reported to be the main predictor of blood pressure in the general population. This was proposed to be because of high resistance artery creatine kinase-BB rapidly regenerating ATP for vascular contractility. Therefore, we assessed whether creatine kinase isoenzyme mRNA levels in human resistance arteries are associated with blood pressure. We isolated resistance-sized arteries from omental fat donated by consecutive women undergoing uterine fibroid surgery. Blood pressure was measured in the sitting position. Vessels of 13 women were included, 6 normotensive and 7 hypertensive, mean age 42.9 years (SE, 1.6) and mean systolic/diastolic blood pressure, 144.8 (8.0)/86.5 (4.3) mm Hg. Arteriolar creatine kinase isoenzyme mRNA was assessed using quantitative real-time polymerase chain reaction. Normalized creatine kinase B mRNA copy numbers, ranging from 5.2 to 24.4 (mean, 15.0; SE, 1.9), showed a near-perfect correlation with diastolic blood pressure (correlation coefficient, 0.9; 95% confidence interval, 0.6–1.0) and were well correlated with systolic blood pressure, with a 90% relative increase in resistance artery creatine kinase B mRNA in hypertensives compared with normotensives, normalized copy numbers were, respectively, 19.3 (SE, 2.0) versus 10.1 (SE, 2.1), P=0.0045. To our knowledge, this is the first direct evidence suggesting that resistance artery creatine kinase mRNA expression levels concur with blood pressure levels, almost doubling with hypertension. These findings add to the evidence that creatine kinase might be involved in the vasculature’s pressor responses.

Meta-Analysis of Placental Transcriptome Data Identifies a Novel Molecular Pathway Related to Preeclampsia

Studies using the placental transcriptome to identify key molecules relevant for preeclampsia are hampered by a relatively small sample size. In addition, they use a variety of bioinformatics and statistical methods, making comparison of findings challenging. To generate a more robust preeclampsia gene expression signature, we performed a meta-analysis on the original data of 11 placenta RNA microarray experiments, representing 139 normotensive and 116 preeclamptic pregnancies. Microarray data were pre-processed and analyzed using standardized bioinformatics and statistical procedures and the effect sizes were combined using an inverse-variance random-effects model. Interactions between genes in the resulting gene expression signature were identified by pathway analysis (Ingenuity Pathway Analysis, Gene Set Enrichment Analysis, Graphite) and protein-protein associations (STRING). This approach has resulted in a comprehensive list of differentially expressed genes that led to a 388-gene meta-signature of preeclamptic placenta. Pathway analysis highlights the involvement of the previously identified hypoxia/HIF1A pathway in the establishment of the preeclamptic gene expression profile, while analysis of protein interaction networks indicates CREBBP/EP300 as a novel element central to the preeclamptic placental transcriptome. In addition, there is an apparent high incidence of preeclampsia in women carrying a child with a mutation in CREBBP/EP300 (Rubinstein-Taybi Syndrome). The 388-gene preeclampsia meta-signature offers a vital starting point for further studies into the relevance of these genes (in particular CREBBP/EP300) and their concomitant pathways as biomarkers or functional molecules in preeclampsia. This will result in a better understanding of the molecular basis of this disease and opens up the opportunity to develop rational therapies targeting the placental dysfunction causal to preeclampsia.