Marie van Dijk

Maternal segregation of the Dutch preeclampsia locus at 10q22 with a new member of the winged helix gene family.

Preeclampsia is a pregnancy-associated disease with maternal symptoms but placental origin. Epigenetic inheritance is involved in some populations. By sequence analysis of 17 genes in the 10q22 region with maternal effects, we narrowed the minimal critical region linked with preeclampsia in the Netherlands to 444 kb. All but one gene in this region, which lies within a female-specific recombination hotspot, encode DNA- or RNA-binding proteins. One gene, STOX1 (also called C10orf24), contained five different missense mutations, identical between affected sisters, cosegregating with the preeclamptic phenotype and following matrilineal inheritance. Four STOX1 transcripts are expressed in early placenta, including invasive extravillus trophoblast, generating three different isoforms. All contain a winged helix domain related to the forkhead (FOX) family. The largest STOX1 isoform has exclusive nuclear or cytoplasmic expression, indicating activation and inactivation, respectively, of the PI3K-Akt-FOX pathway. Because all 38 FOX proteins and all 8 STOX1 homologs have either tyrosine or phenylalanine at position 153, the predominant Y153H variation is highly mutagenic by conservation criteria but subject to incomplete penetrance. STOX1 is a candidate for preeclampsia controlling polyploidization of extravillus trophoblast.

Genetics of preeclampsia: paradigm shifts

Segregation of preeclampsia into early-onset, placental and late-onset, maternal subtypes along with the acknowledgement of the contribution of epigenetics in placentally expressed genes proved to be a key first step in the identification of essential gene variants associated with preeclampsia. Application of this insight to other populations and related pregnancy-induced syndromes, such as HELLP, and acknowledgment of the features shared between chromosomal loci associated with preeclampsia in different populations provide the rationale for new strategies for the identification of susceptibility genes and for new and more effective diagnostic strategies.

HELLP babies link a novel lincRNA to the trophoblast cell cycle

The HELLP syndrome is a pregnancy-associated disease inducing hemolysis, elevated liver enzymes, and low platelets in the mother. Although the HELLP symptoms occur in the third trimester in the mother, the origin of the disease can be found in the first trimester fetal placenta. A locus for the HELLP syndrome is present on chromosome 12q23 near PAH. Here, by multipoint nonparametric linkage, pedigree structure allele sharing, and haplotype association analysis of affected sisters and cousins, we demonstrate that the HELLP locus is in an intergenic region on 12q23.2 between PMCH and IGF1. We identified a novel long intergenic noncoding RNA (lincRNA) transcript of 205,012 bases with (peri)nuclear expression in the extravillous trophoblast using strand-specific RT-PCR complemented with RACE and FISH. siRNA-mediated knockdown followed by RNA-sequencing, revealed that the HELLP lincRNA activated a large set of genes that are involved in the cell cycle. Furthermore, blocking potential mutation sites identified in HELLP families decreased the invasion capacity of extravillous trophoblasts. This is the first large noncoding gene to be linked to a Mendelian disorder with autosomal-recessive inheritance.

Differential activation of murine herpesvirus 68- and Kaposi's sarcoma-associated herpesvirus-encoded ORF74 G protein-coupled receptors by human and murine chemokines

ABSTRACT Infection of mice with murine gammaherpesvirus 68 (MHV-68) is a well-characterized small animal model for the study of gammaherpesvirus infection. MHV-68 belongs to the same herpesvirus family as herpesvirus saimiri (HVS) of New World squirrel monkeys and human herpesvirus 8 (HHV-8) (also referred to as Kaposis sarcoma-associated herpesvirus [KSHV]). The open reading frame ORF74 of HVS, KSHV, and MHV-68 encodes a protein with homology to G protein-coupled receptors and chemokine receptors in particular. ORF74 of KSHV (human ORF74 [hORF74]) is highly constitutively active and has been implicated in the pathogenesis of Kaposis sarcoma. MHV-68-encoded ORF74 (mORF74) is oncogenic and has been implicated in viral replication and reactivation from latency. Here, we show that mORF74 is a functional chemokine receptor. Chemokines with an N-terminal glutamic acid-leucine-arginine (ELR) motif (e.g., KC and macrophage inflammatory protein 2) act as agonists on mORF74, activating phospholipase C, NF-κB, p44/p42 mitogen-activated protein kinase, and Akt signaling pathways and inhibiting formation of cyclic AMP. Using 125I-labeled CXCL1/growth-related oncogene α as a tracer, we show that murine CXCL10/gamma interferon-inducible protein 10 binds mORF74, and functional assays show that it behaves as an antagonist for this virally encoded G protein-coupled receptor. Profound differences in the upstream activation of signal transduction pathways between mORF74 and hORF74 were found. Moreover, in contrast to hORF74, no constitutive activity of mORF74 could be detected.

The Pre-Eclampsia Gene STOX1 Controls a Conserved Pathway in Placenta and Brain Upregulated in Late-Onset Alzheimer's Disease

Pre-eclampsia and late-onset Alzheimers disease (LOAD) share no clinical features. In contrast to these clinical dissimilarities, striking parallels exist between the (epi)genetic features associated with pre-eclampsia and LOAD for the genes located on 10q22. The parallels in identity between the 10q22 genes involved and active in the organs (placenta, brain) primarily affected in the respective diseases led us to explore, if the pre-eclampsia susceptibility gene STOX1 is functionally involved in LOAD. We demonstrate that isoform A of STOX1 is abundantly expressed in the brain, correlates with severity of disease, and selectively transactivates LRRTM3 in neural cells with increased amyloid-beta protein precursor processing. Similar in vitro results were seen in trophoblast. Our data indicate that STOX1 controls a conserved pathway shared between placenta and brain with overexpression in LOAD.

STOX1: Key player in trophoblast dysfunction underlying early onset preeclampsia with growth retardation.

Currently, only two preeclampsia susceptibility genes (ACVR2A, STOX1) have been identified within confirmed regions with significant genome-wide linkage, although many genetic screens in multiple populations have been performed. In this paper, we focus on the STOX1 gene. The epigenetic status of this gene is discussed explaining the maternal transmission of the STOX1 susceptibility allele observed in preeclamptic families. The known upstream regulation and downstream effector genes of the transcription factor STOX1 are described. Finally, we propose a model in which we combine the cell type-specific and allele-specific effects of STOX1. This includes intrinsic effects (differential CpG island methylation) and extrinsic effects (regulation of effector genes).

Helix 8 of the Viral Chemokine Receptor ORF74 Directs Chemokine Binding

The constitutively active G-protein-coupled receptor and viral oncogene ORF74, encoded by Kaposi sarcoma-associated herpesvirus (human herpesvirus 8), binds a broad range of chemokines, including CXCL1 (agonist), CXCL8 (neutral ligand), and CXCL10 (inverse agonist). Although chemokines interact with the extracellular N terminus and loops of the receptor, we demonstrate that helix 8 (Hx8) in the intracellular carboxyl tail (C-tail) of ORF74 directs chemokine binding. Partial deletion of the C-tail resulted in a phenotype with reduced constitutive activity but intact regulation by ligands. Complete deletion of the C-tail, including Hx8, resulted in an inactive phenotype that lacks CXCL8 binding sites and has an increased number of binding sites for CXCL10. Similar effects were obtained with the single R7.61322W or Q7.62323P mutations in Hx8. We propose that the conserved charged or polar side chain at position 7.61 has a specific role in stabilizing the end of transmembrane domain 7 (TM7). Disruption of Hx8 by deletion or mutation distorts an H-bonding network, involving highly conserved amino acids within TM2, TM7, and Hx8, that is crucial for positioning of the TM domains, coupling to Gαq, and CXCL8 binding. Thus, Hx8 appears to exert a key role in receptor stabilization through the conserved residue R7.61, directing the ligand binding profile of ORF74 and likely also that of other class A G-protein-coupled receptors.

(Epi)genetics of pregnancy-associated diseases.

This review describes the current knowledge regarding genetics and epigenetics of pregnancy-associated diseases with placental origin. We discuss the effect on genetic linkage analyses when the fetal genotype determines the maternal phenotype. Secondly, the genes identified by genome-wide linkage studies to be associated with pre-eclampsia (ACVR2A, STOX1) and the HELLP-syndrome (LINC-HELLP) are discussed regarding their potential functions in the etiology of disease. Furthermore, susceptibility genes identified by candidate gene approaches (e.g., CORIN) are described. Next, we focus on the additional challenges that come when epigenetics also play a role in disease inheritance. We discuss the maternal transmission of the chromosome 10q22 pre-eclampsia linkage region containing the STOX1 gene and provide further evidence for the role of epigenetics in pre-eclampsia based on the cdkn1c mouse model of pre-eclampsia. Finally, we provide recommendations to unravel the genetics of pregnancy-associated diseases, specifically regarding clear definitions of patient groups and sufficient patient numbers, and the potential usefulness of (epi)genetic data in early non-invasive biomarker development.

Differential methylation of STOX1 in human placenta.

The 10q22 chromosomal region with genomic linkage to pre-eclampsia in Dutch females shows a parent-of-origin effect with maternal transmission of the Y153H susceptibility allele of the STOX1 gene. Although the CpG island within the STOX1 promoter region shows no differential methylation, this study describes the identification of a differentially methylated region (DMR) in intron 1 of the STOX1 gene. Methylation coincides with STOX1 expression, where high methylation leads to reduced expression. In the SGHPL-5 extravillous trophoblast cell line allele-specific expression was observed in a subset of cells. Although no allele-specific expression could be detected in early placenta samples, these samples did show an increase in methylation when they were homozygous for the Y153H susceptibility allele. Allele-specific methylation was observed in column extravillous trophoblast samples with the methylated allele being paternal in origin. We conclude that STOX1 is paternally imprinted, maternally expressed, with the DMR identified in this study showing parental-specific methylation in specific cell-types, hypothesized to occur in villous cytotrophoblasts, and proven in column extravillous trophoblasts originating from the anchoring villus. In other (placental) cells methylation is independent of parental origin, but regulates STOX1 expression with the Y153H genotype directing the level of methylation.

Naturally occurring variation in trophoblast invasion as a source of novel (epigenetic) biomarkers

During the first trimester of pregnancy fetal trophoblasts invade the maternal decidua, thereby remodeling the maternal spiral arteries. This process of trophoblast invasion is very similar to cancer cell invasion, with multiple signaling pathways shared between the two. Pregnancy-related diseases, e.g., pre-eclampsia, and cancer metastasis start with a decrease or increase in cellular invasion, respectively. Here, we investigate if first trimester placental explants can be used to identify epigenetic factors associated with changes in cellular invasion and their potential use as biomarkers. We show that the outgrowth potential of first trimester explants significantly correlates with promoter methylation of PRKCDBP and MMP2, two genes known to be differentially methylated in both placenta and cancer. The increase in methylation percentage of placental cells coincides with an increase in invasion potential. Subsequently, as a non-invasive marker must be detectable in blood, plasma samples of pregnant and non-pregnant women were analyzed. The MMP2 promoter showed high methylation levels in non-pregnant plasma samples, which decreased in pregnant plasma samples which also contain placental DNA. The decrease in methylated plasma DNA during pregnancy is most likely due to the fractional increase in unmethylated placental DNA. This suggests that the level of unmethylated DNA has the potential to be used as an invasion marker, where higher levels of unmethylated DNA indicate a lower invasion potential of trophoblasts. These proof of principle data provide evidence that human first trimester placental explants are an excellent ex vivo model system to identify (epigenetic) factors and thus potential biomarkers associated with changes in cellular invasion, e.g., to detect pregnancy-related diseases or cancer metastasis. To identify novel biomarkers the next step is to correlate naturally occurring variation in invasion potential to changes in (epigenetic) factors by genome-wide approaches such as massively parallel sequencing.