ACVR2A promoter polymorphism rs1424954 in the Activin-A signaling pathway in trophoblasts.

INTRODUCTION: Pre-eclampsia is a pregnancy-specific disorder and characterized by reduced trophoblast invasion and reduced spiral artery remodeling in the first trimester placenta. A polymorphism located in the promoter region of ACVR2A (rs1424954 (A > G)) has previously been shown to be significantly associated with pre-eclampsia. METHODS: The effects of this variant on ACVR2A expression and its function in the Activin-A signaling pathway were studied by transfections in SGHPL-5 extravillous trophoblasts followed by qRT-PCR. RESULTS: Here we show that the ACVR2A promoter susceptibility variant causes a downregulation of ACVR2A expression. We also provide evidence for transcription of a so-called PROMPT (PROMoter-uPstream-Transcript) in the opposite direction of ACVR2A, containing the polymorphism, and downregulated when the susceptibility allele is carried, which either shares the same promoter as ACVR2A or is a non-coding RNA that is able to enhance ACVR2A transcription. Furthermore, when the effect of the susceptibility variant is mimicked by knockdown of ACVR2A, physiologic concentrations of Activin-A cause a reduction in NODAL mRNA expression in the SGHPL-5 trophoblasts, indicative of a protective effect as reduction in NODAL expression is associated with an increase in trophoblast invasion. However, at pathologic levels of Activin-A, as found in pre-eclampsia, this effect is no longer seen, and we show this is potentially caused by a lack of downregulation of ACVR2B. DISCUSSION: The combined data suggest a double hit phenomenon in which the first hit, the promoter variant, together with the second hit, pathological levels of Activin-A, lead to high levels of NODAL, associated with reduced trophoblast invasion and observed in pre-eclamptic placentas.

IFPA meeting 2014 workshop report: Animal models to study pregnancy pathologies; new approaches to study human placental exposure to xenobiotics; biomarkers of pregnancy pathologies; placental genetics and epigenetics; the placenta and stillbirth and fetal growth restriction.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2014 there were six themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of animal models, xenobiotics, pathological biomarkers, genetics and epigenetics, and stillbirth and fetal growth restriction.

Relevance of IL7R genotype and mRNA expression in Dutch patients with multiple sclerosis.

BACKGROUND: The interleukin 7 receptor (IL7R) has been recognized as a susceptibility gene for Multiple Sclerosis (MS). Analysis of rs6897932 (the most strongly MS-associated single nucleotide polymorphism (SNP)), showed effects of genotype on the relative expression of membrane-bound to total amount of IL7R mRNA. OBJECTIVE: We assessed the relevance of IL7R on MS phenotype (including clinical and magnetic resonance imaging (MRI) parameters) at DNA and mRNA level in Dutch patients with MS. METHODS: The genotype of rs6897932 was analyzed in 697 patients with MS and 174 healthy controls. The relevance of genotype and carriership of the C allele on MS phenotype (disease activity and severity, using clinical and MRI parameters) was assessed. In addition, relative gene expression of membrane-bound to total IL7R mRNA was analyzed with respect to disease phenotype in a subgroup of 95 patients with early relapsing MS. RESULTS: In particular, homozygosity for the risk allele is a risk factor for MS in our population (OR(CC vs CT and TT) = 1.65 (95% CI: 1.18-2.30), two-sided p = 0.004). However, no effect of genotype or the relative expression of membrane-bound IL7R (presence of exon 6-7) to total amount of IL7R mRNA (presence of exon 4-5) was found on MS phenotype. DISCUSSION: Homozygosity for the IL7R exon 6 rs6897932 C allele is associated with a higher risk for MS in our Dutch population. No effect was found of genotype or mRNA expression on disease phenotype.

Spontaneous MxA mRNA level predicts relapses in patients with recently diagnosed MS.

OBJECTIVE: To determine if myxovirus resistance protein A (MxA) mRNA is related to clinical disease activity in multiple sclerosis (MS). METHODS: Baseline MxA mRNA levels were measured in a prospective cohort of 116 untreated patients with early MS and were related to clinical relapses and MRI at baseline and at follow-up. RESULTS: Low levels of MxA mRNA were associated with the occurrence of relapses (p = 0.002) and contrast-enhancing lesions (CELs) on baseline MRI (p = 0.045). In addition, high baseline MxA mRNA levels were related to a longer time to a first new relapse (hazard ratio [HR] 0.59; 95% confidence interval [CI] 0.35-1.00; p = 0.044). Adding the absence of CELs to high MxA mRNA, the predictive value increased (HR 0.35; 95% CI 0.17-0.74; p = 0.006), clearly showing a cumulative value for combining both factors. CONCLUSIONS: MxA mRNA is related to clinical exacerbations, the number of CELs on MRI, and is indicative for the time to a subsequent relapse. If confirmed, MxA mRNA has potential as a biomarker for clinical disease activity in MS.

Lack of interferon-beta bioactivity is associated with the occurrence of relapses in multiple sclerosis.

BACKGROUND: Treatment failure to Interferon-beta (IFNbeta) in multiple sclerosis (MS) can only partly be explained by anti-IFNbeta neutralising antibodies (NAb). Myxovirus resistance protein A (MxA) mRNA, reflecting IFNbeta bioactivity, is studied as an alternative biomarker for IFNbeta therapy response. Although absent IFNbeta bioactivity is associated with NAb and NAb are associated with reduced drug efficacy, the direct relationship between IFNbeta bioactivity and clinical disease activity is largely unknown. METHODS: We enrolled 126 consecutive relapsing-remitting MS patients on IFNbeta treatment. MxA mRNA expression was assessed 4 h after IFNbeta injection. Biological response status was determined after 3 months, by combined measurement of MxA mRNA expression and induction (MxA mRNA expression after/before IFNbeta injection). Patients were considered biological non-responders when both MxA mRNA expression and MxA mRNA induction were negative. RESULTS: Biological non-responders showed a significantly higher annualised relapse rate and smaller proportion relapse-free patients compared with biological responders (relapse rate 0.81 vs. 0.37; proportion relapse free 37% vs. 67%). CONCLUSIONS: Our results suggest that a lack of IFNbeta bioactivity is associated with the occurrence of relapses and therefore can be useful as a biomarker for unresponsiveness to IFNbeta.

Interferon-beta bioactivity measurement in multiple sclerosis: feasibility for routine clinical practice.

BACKGROUND: Neutralising antibodies (NAb) to interferon beta (IFN beta) are associated with a reduced bioactivity and efficacy of IFN beta in multiple sclerosis (MS). Unclear is how to apply IFN beta bioactivity measurements (quantification of Myxovirus resistance protein A (MxA) mRNA) in clinical practice. OBJECTIVES: To evaluate value and feasibility of IFN beta bioactivity measurement with a single MxA mRNA measurement for screening and a second measurement before and after IFN beta administration for definite confirmation of IFN beta bioactivity status. METHODS: In 79 MS patients MxA mRNA expression was determined 4 hours after IFN beta administration. If inadequate, MxA mRNA expression testing was repeated 3 months afterwards, comparing post- and pre injection samples to determine whether IFNb bioactivity was persistently lacking. MxA mRNA expression was compared to NA beta titres, determined by the cytopathic effect assay (CPE). RESULTS: NAb titres correlated significantly with MxA mRNA expression and MxA mRNA induction. Of all screened patients, only one patient had adequate MxA mRNA expression and high NAb titres simultaneously. Of the biological non-responders at second measurement (21/55), 17 (81%) were high-titre NAb positive, 1 (5%) was low-titre NAb positive and 3 (14%) were NAb negative. Without considering the pre-injection measurement, two more NAb negative patients would have tested negative for IFN beta bioactivity, emphasizing the need of a pre-injection sample. CONCLUSIONS: Our data suggest that for IFN beta bioactivity screening a single post-injection measurement seems reasonable. However, MxA induction measurement based on both pre- and post-IFN beta injection samples at second measurement is somewhat more precise in determining ultimate IFN beta bioactivity status.

Placental gene expression and pre-eclampsia.

Pre-eclampsia and related syndromes, such as the HELLP (haemolysis, elevated liver enzymes and low platelets) syndrome, have a strong genetic component, although the nature, origin and recurrence rate of the genetic risks involved are not necessarily the same for each form of pregnancy-induced hypertension or population. This review addresses these items in the context of the recent understanding that (a) common diseases can be caused by common genetic variations, (b) the placental genotype can control the maternal phenotype, (c) epistasis and epigenetics are involved, and (d) a conserved pathway shared between organs is possible as well as a common, pathophysiological pathway is shared between syndromes.

Basic helix-loop-helix transcription factor profiling of lung tumors shows aberrant expression of the proneural gene atonal homolog 1 (ATOH1, HATH1, MATH1) in neuroendocrine tumors.

Microarray-based expression profiling studies of lung adenocarcinomas have defined neuroendocrine subclasses with poor prognosis. As neuroendocrine development is regulated by members of the achaete-scute and atonal classes of basic helix-loop-helix (bHLH) transcription factors, we analyzed lung tumors for expression of these factors. Out of 13 bHLH genes tested, 4 genes, i.e., achaete-scute complex-like 1 (ASCL1, HASH1, Mash1), atonal homolog 1 (ATOH1, HATH1, MATH1), NEUROD4 (ATH-3, Atoh3, MATH-3) and neurogenic differentiation factor 1 (NEUROD1, NEUROD, BETA2), showed differential expression among lung tumors and absent or low expression in normal lung. As expected, tumors that have high levels of ASCL1 also express neuroendocrine markers, and we found that this is accompanied by increased levels of NEUROD1. In addition, we found ATOH1 expression in 9 (16%) out of 56 analyzed adenocarcinomas and these tumors showed neuroendocrine features as shown by dopa decarboxylase mRNA expression and immunostaining for neuroendocrine markers. ATOH1 expression as well as NEUROD4 was observed in small cell lung carcinoma (SCLC), a known neuroendocrine tumor. Since ATOH1 is not known to be involved in normal lung development, our results suggest that aberrant activation of ATOH1 leads to a neuroendocrine phenotype similar to what is observed for ASCL1 activation during normal neuroendocrine development and in lung malignancies. Our preliminary data indicate that patients with ATOH1-expressing adenocarcinomas might have a worse prognosis.

C21ORF105, A chromosome 21-encoded mRNA, is not a discriminative marker gene for prediction of Down syndrome in maternal plasma.

OBJECTIVES: The presence and detectability of placental mRNA in maternal plasma opens possibilities for the development of non-invasive prenatal diagnostic tests. In this study, we tested C21orf105, a chromosome 21-encoded, placentally expressed gene, in maternal plasma of women carrying a fetus with or without trisomy 21. METHODS: Using real-time RT-PCR, we determined transcript levels of target (C21orf105) and reference (hPL) genes in first-trimester plasma samples. Plasma was obtained from first-trimester EDTA blood after two sequential centrifugation steps and stored at -70 degrees C. After RNA extraction, quantitative RT-PCR was performed using Taqman probes. RESULTS: From the 51 samples, 43 samples were conclusive. Comparison of transcript levels of C21orf105 in both groups showed no significant differences. When expressed as ratios of hPL/C21orf105, the differences between trisomy 21 and normal pregnancies remained non-significant. CONCLUSIONS: The amount of C21orf105 mRNA in maternal plasma, although situated in the Down syndrome critical region on chromosome 21 and up regulated in trisomy 21 placentas, is not higher in women carrying a fetus with trisomy 21.

Elevated C-reactive protein levels during first trimester of pregnancy are indicative of preeclampsia and intrauterine growth restriction.

C-reactive protein (CRP) is a marker of tissue damage and inflammation. Maternal levels of CRP are elevated in overt preeclampsia, but there is still debate about its use as a predictive marker for preeclampsia during the first and second trimesters of pregnancy. In this study, we measured CRP levels during the first trimester of pregnancy in women who later developed preeclampsia or gave birth to a growth-restricted baby. In total, 107 women from a low-risk population participated in the study, six women developed preeclampsia and nine gave birth to a growth-restricted baby. Although there is a large overlap in measured CRP levels between the three groups, mean CRP levels were significantly elevated in women who later developed preeclampsia (P=0.031) or delivered a growth-restricted baby (P=0.041) when compared with women from the control group, matched for maternal and gestational age, parity, and gravidity. This study shows that in a low-risk population, CRP levels are already elevated between weeks 10 and 14 in pregnant women who develop preeclampsia or deliver a growth-restricted baby.

Identification of novel mutations in MLC1 responsible for megalencephalic leukoencephalopathy with subcortical cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an inherited neurologic disorder with macrocephaly before the age of one and slowly progressive deterioration of motor functions. Magnetic resonance imaging shows diffusely abnormal and swollen white matter of the cerebral hemispheres and the presence of subcortical cysts in the anterior-temporal region and often also in the frontoparietal region. Mutations in the MLC1 gene, encoding a putative membrane protein, have been recently identified as a cause for MLC. Here, we describe 14 new mutations in 18 patients. Two identified polymorphisms lead to alterations of amino acid residues. The role, suggested by others, of a mutation in the MLC1gene in catatonic schizophrenia and the possible function of the MLC1 protein as a cation channel are discussed.

Subunits of the translation initiation factor eIF2B are mutant in leukoencephalopathy with vanishing white matter.

Leukoencephalopathy with vanishing white matter (VWM) is an inherited brain disease that occurs mainly in children. The course is chronic-progressive with additional episodes of rapid deterioration following febrile infection or minor head trauma. We have identified mutations in EIF2B5 and EIF2B2, encoding the epsilon- and beta-subunits of the translation initiation factor eIF2B and located on chromosomes 3q27 and 14q24, respectively, as causing VWM. We found 16 different mutations in EIF2B5 in 29 patients from 23 families. We also found two distantly related individuals who were homozygous with respect to a missense mutation in EIF2B2, affecting a conserved amino acid. Three other patients also had mutations in EIF2B2. As eIF2B has an essential role in the regulation of translation under different conditions, including stress, this may explain the rapid deterioration of people with VWM under stress. Mutant translation initiation factors have not previously been implicated in disease.

Plasma placenta growth factor levels in midtrimester pregnancies.

OBJECTIVE: Previous studies have shown decreased levels of placenta growth factor in serum of pregnant women with preeclampsia. The aim of this study was to investigate whether levels of placenta growth factor are decreased before the clinical onset of preeclampsia, and whether placenta growth factor levels are decreased in pregnancies complicated by intrauterine growth restriction. METHODS: From an ongoing longitudinal study, 101 plasma samples were collected from 72 pregnant women at weeks 11-21 of gestation. Placenta growth factor levels were determined retrospectively in plasma using an enzyme-linked immunosorbent assay. Correlations between plasma concentrations of placenta growth factor and pregnancy outcome were evaluated. RESULTS: Plasma samples of 72 patients were analyzed. Forty-four patients had no pregnancy complications, 18 developed preeclampsia, and 10 women had pregnancies complicated by intrauterine growth restriction. Between week 17 and week 21 of pregnancy, a significantly lower level of placenta growth factor was found in plasma of patients who later developed preeclampsia (n = 10), compared with control pregnancies (n = 25, P = .004). In women with a growth-restricted baby at birth (n = 5), levels of placenta growth factor were also low. CONCLUSIONS: Our results show that plasma placenta growth factor levels are decreased before preeclampsia is clinically evident. The data suggest that placenta growth factor may be useful to determine the relative risk of developing preeclampsia and intrauterine growth restriction.

Decreased galanin mRNA levels in growth hormone-releasing hormone neurons after perinatally induced growth retardation.

Intrauterine growth retardation (IUGR) is associated with persistent postnatal growth retardation accompanied by dysfunction of the hypothalamic components of the growth hormone (GH) axis. At the adult stage, this is reflected by increased somatostatin (SS) and decreased neuropeptide Y (NPY) mRNA levels, whereas the GH-releasing hormone (GHRH) mRNA levels are normal and the output of GH remains unchanged. To extend our insight into the hypothalamic control of GH secretion in growth retarded rats, we determined galanin (GAL) mRNA levels at the adult stage of perinatally malnourished (i.e. IUGR and early postnatally food restricted) rats. Analyses included comparison of GAL mRNA levels in GHRH neurons in perinatally malnourished adult rats using a semi-quantitative double labeling in situ hybridization technique. We report that IUGR is accompanied by a 60% decrease in GAL mRNA levels in all GHRH neurons in the male IUGR group whereas a tendency towards a decrease was observed in the male early postnatally food restricted (FR) group. These effects became more pronounced when the analysis was restricted to GHRH neurons coexpressing GAL mRNA i.e. decreased GAL mRNA levels were seen in both male and female IUGR rats and in FR males. These data show that GAL mRNA levels in GHRH neurons are persistently decreased after perinatal malnutrition. Taking these results together with our previous data on SS, NPY and GHRH mRNA levels, we can conclude that IUGR leads to a reprogramming of the hypothalamic regulation of GH secretion.

Absence of androgen receptor in the growth hormone releasing hormone-containing neurones in the rat mediobasal hypothalamus.

Growth hormone (GH) secretory patterns are influenced by gonadal steroids, at least in part, through modulation of hypothalamic somatostatin and GH releasing hormone (GHRH) secretion. In the adult male rat, testosterone appears to stimulate somatostatin gene expression by acting directly on androgen receptors in somatostatin neurones. The mechanism by which gonadal status influences hypothalamic GHRH gene expression is less clear. Gonadectomy reduces GHRH mRNA expression in rats, and this reduction can be prevented by the administration of testosterone or partly by a nonaromatizable androgen. While these observations suggest that androgen receptors mediate the actions of gonadal steroids on GHRH gene expression, they do not provide any information about the location of the androgen receptors involved in this process. To determine whether GHRH neurones themselves express androgen receptors, we double immunolabelled hypothalamic sections from colchicine-pretreated male rats. Although there was an overlap in the anatomical distribution of GHRH and androgen receptor-containing cell bodies, none of the nearly 900 GHRH immunolabelled cells we examined in each mediobasal hypothalamus appeared to contain androgen receptors. These results suggest that GHRH-expressing neurones are not direct targets for androgens and therefore the effects of testosterone on GHRH gene expression must be produced indirectly by some other neural or endocrine intermediary process.

The Human Achaete Scute Homolog 2 gene contains two promotors, generating overlapping transcripts and encoding two proteins with different nuclear localization.

Placental development involves control by the basic helix-loop-helix transcription factor Mash2. Transcript analysis of the Human Achaete Scute Homolog 2 (HASH2) mRNA revealed the presence of two overlapping transcripts in first trimester placentae. The two transcripts (2.6 and 1.5 kb) are generated by two promotors which are separated by 1.1 kb, generating transcripts 1 and 2, respectively. Surprisingly, in transcript 1 which shows a broad expression, a second potential coding region, tentatively called Human Achaete Scute Associated Protein (HASAP) was present. Transcript 2 contains the HASH2 encoding region only. Analysis of protein expression from both transcripts by transfection studies with eGFP fusion proteins, revealed that both coding regions are translated from their endogenous translation initiation site and showed that both proteins are transported to the nucleus. HASH2 is distributed throughout the nucleus but the HASAP protein is transported into nuclear compartments, the nucleoli. In addition, the HASAP protein lacks the bHLH domain and bears no homology to known proteins. Moreover, allele-specific RT-PCR showed the human gene not to be subject to imprinting, possibly reflecting the biallelic expression of one of both transcripts. Our data indicate a species-specific difference between mouse and human expression of the Achaete Scute Homolog 2 and suggests a dual function of the human homologue.

Allelic IGF2R repression does not correlate with expression of antisense RNA in human extraembryonic tissues.

In the mouse, expression of an antisense Igf2r RNA (Air) is correlated with Igf2r repression on the paternal allele. One of the possible models for Igf2r repression could be through promoter competition or through the action of the Air RNA, in, e.g., transcriptional interference or repressor binding. These models predict the conservation of AIR RNA in human samples with monoallelic IGF2R expression and the production of AIR RNA in first-trimester human tissues. However, by strand-specific RT-PCR and by ribonuclease protection assay we have not detected any AIR RNA in first-trimester placental tissue samples, not even in samples that downregulate IGF2R expression in an allele-specific manner. This indicates that in contrast to the mouse, allelic IGF2R repression in the developing human placenta does not correlate with AIR expression.

HLA-G expression in trophoblast cells circulating in maternal peripheral blood during early pregnancy.

OBJECTIVE: The aim of this study was to assess the use of circulating trophoblast cells in maternal peripheral blood for noninvasive prenatal diagnosis of numeric chromosomal aberrations. STUDY DESIGN: A combined procedure for immunocytochemical identification and deoxyribonucleic acid fluorescence in situ hybridization was used after a single enrichment step consisting of density gradient centrifugation. A specific HLA-G monoclonal antibody was used in combination with X and Y chromosome specific probes in deoxyribonucleic acid fluorescence in situ hybridization to confirm fetal identity of cells bearing HLA-G in the case of a male fetus. RESULTS: We detected fetal trophoblast cells expressing HLA-G in maternal blood starting at 9 weeks' gestation. In addition to fetal sex prediction with X and Y chromosome-specific probes, fetal aneuploidy was confirmed in peripheral blood from a pregnancy complicated by trisomy 21. CONCLUSION: Although the numbers of fetal cells were extremely low, the proof of concept was demonstrated. Early noninvasive prenatal screening for numeric chromosomal abnormalities with fetal trophoblast cells is feasible.

Mutations of MLC1 (KIAA0027), encoding a putative membrane protein, cause megalencephalic leukoencephalopathy with subcortical cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal recessive disorder characterized by macrocephaly, deterioration of motor functions with ataxia, and spasticity, eventuating in mental decline. The brain appears swollen on magnetic resonance imaging, with diffuse white-matter abnormalities and the invariable presence of subcortical cysts. MLC was recently localized on chromosome 22q(tel). We have narrowed down the critical region by linkage analysis of 11 informative families with MLC to a region of approximately 250 kb, containing four known genes. One family with two patients who were siblings did not display linkage between the MLC phenotype and any of the analyzed microsatellite markers on chromosome 22q(tel), suggesting genetic heterogeneity and the existence of at least a second MLC locus. The maximum two-point LOD score for the 11 families was 6.6 at recombination fraction .02. Twelve different mutations in seven informative and six uninformative families were found in one of the candidate genes, KIAA0027, which we renamed "MLC1." The gene encodes a putative membrane protein with eight predicted transmembrane domains. The patients of one family were compound heterozygotes for mutations that both introduced stop codons. The mutations further included frameshifts, splice-acceptor mutations, a putative splice-donor mutation, and amino acid substitutions of residues in predicted transmembrane domains. These data provide strong evidence that mutations of MLC1 cause the disease.

Persistent changes in somatostatin and neuropeptide Y mRNA levels but not in growth hormone-releasing hormone mRNA levels in adult rats after intrauterine growth retardation.

A reduction in the availability of oxygen and nutrients across the placenta in the last trimester of pregnancy may lead to intrauterine growth retardation (IUGR) which, in turn, may cause a persistent postnatal growth failure. However, it is unknown whether this persistent growth retardation is centrally mediated through alterations in the components of the growth hormone (GH)-axis. We tested the hypothesis that alterations in the development of the central components of the GH-axis contribute to the persistent growth failure observed after experimentally induced IUGR or early postnatal food restriction (FR) in the rat. Using semi-quantitative in situ hybridization, we compared somatostatin (SS), GH-releasing hormone (GHRH) and neuropeptide Y (NPY) mRNA levels in adult rats experimentally subjected to IUGR or FR. We report that IUGR increased the expression of SS mRNA in the periventricular nucleus (PeN) of adult male and female rats by 128% and 153% respectively, did not alter the expression of GHRH mRNA in the arcuate nucleus (ARC) and decreased the NPY mRNA expression in the ARC by 73% in males and 61% in females, whereas in the FR group no changes in the expression of these mRNAs were observed. These data show that the timing of malnutrition or the presence of the placenta is important for the long-term alterations since the effects only occurred in the prenatally induced growth retardation and not in the early postnatally induced growth retardation group.