The application of epiphenotyping approaches to DNA methylation array studies of the human placenta.

BACKGROUND: Genome-wide DNA methylation (DNAme) profiling of the placenta with Illumina Infinium Methylation bead arrays is often used to explore the connections between in utero exposures, placental pathology, and fetal development. However, many technical and biological factors can lead to signals of DNAme variation between samples and between cohorts, and understanding and accounting for these factors is essential to ensure meaningful and replicable data analysis. Recently, "epiphenotyping" approaches have been developed whereby DNAme data can be used to impute information about phenotypic variables such as gestational age, sex, cell composition, and ancestry. These epiphenotypes offer avenues to compare phenotypic data across cohorts, and to understand how phenotypic variables relate to DNAme variability. However, the relationships between placental epiphenotyping variables and other technical and biological variables, and their application to downstream epigenome analyses, have not been well studied. RESULTS: Using DNAme data from 204 placentas across three cohorts, we applied the PlaNET R package to estimate epiphenotypes gestational age, ancestry, and cell composition in these samples. PlaNET ancestry estimates were highly correlated with independent polymorphic ancestry-informative markers, and epigenetic gestational age, on average, was estimated within 4 days of reported gestational age, underscoring the accuracy of these tools. Cell composition estimates varied both within and between cohorts, as well as over very long placental processing times. Interestingly, the ratio of cytotrophoblast to syncytiotrophoblast proportion decreased with increasing gestational age, and differed slightly by both maternal ethnicity (lower in white vs. non-white) and genetic ancestry (lower in higher probability European ancestry). The cohort of origin and cytotrophoblast proportion were the largest drivers of DNAme variation in this dataset, based on their associations with the first principal component. CONCLUSIONS: This work confirms that cohort, array (technical) batch, cell type proportion, self-reported ethnicity, genetic ancestry, and biological sex are important variables to consider in any analyses of Illumina DNAme data. We further demonstrate the specific utility of epiphenotyping tools developed for use with placental DNAme data, and show that these variables (i) provide an independent check of clinically obtained data and (ii) provide a robust approach to compare variables across different datasets. Finally, we present a general framework for the processing and analysis of placental DNAme data, integrating the epiphenotype variables discussed here.

eoPred: predicting the placental phenotype of early-onset preeclampsia using public DNA methylation data.

Background: A growing body of literature has reported molecular and histological changes in the human placenta in association with preeclampsia (PE). Placental DNA methylation (DNAme) and transcriptomic patterns have revealed molecular subgroups of PE that are associated with placental histopathology and clinical phenotypes of the disease. However, the clinical and molecular heterogeneity of PE both across and within subtypes complicates the study of this disease. PE is most strongly associated with placental pathology and adverse fetal and maternal outcomes when it develops early in pregnancy. We focused on placentae from pregnancies affected by preeclampsia that were delivered before 34 weeks of gestation to develop eoPred, a predictor of the DNAme signature associated with the placental phenotype of early-onset preeclampsia (EOPE). Results: Public data from 83 placental samples (HM450K), consisting of 42 EOPE and 41 normotensive preterm birth (nPTB) cases, was used to develop eoPred-a supervised model that relies on a highly discriminative 45 CpG DNAme signature of EOPE in the placenta. The performance of eoPred was assessed using cross-validation (AUC = 0.95) and tested in an independent validation cohort (n = 49, AUC = 0.725). A subset of fetal growth restriction (FGR) and late-PE cases showed a similar DNAme profile at the 45 predictive CpGs, consistent with the overlap in placental pathology between these conditions. The relationship between the EOPE probability generated by eoPred and various phenotypic variables was also assessed, revealing that it is associated with gestational age, and it is not driven by cell composition differences. Conclusion: eoPred relies on a 45-CpG DNAme signature to predict a homogeneous placental phenotype of EOPE in a discrete or continuous manner. Using this classifier should 1) aid in the study of placental insufficiency and improve the consistency of future placental DNAme studies of PE, 2) facilitate identifying the placental phenotype of EOPE in public data sets and 3) importantly, standardize the placental diagnosis of EOPE to allow better cross-cohort comparisons. Lastly, classification of cases with eoPred will be useful for investigating the relationship between placental pathology and genetic or environmental variables.

A de novo mosaic mutation in SPAST with two novel alternative alleles and chromosomal copy number variant in a boy with spastic paraplegia and autism spectrum disorder.

Here we report a 12 year old male with an extreme presentation of spastic paraplegia along with autism and dysmorphisms. Whole exome sequencing identified a predicted pathogenic pair of missense variants in SPAST at the same chromosomal location, each with a different alternative allele, while a chromosome microarray identified a 1.73 Mb paternally inherited copy gain of 1q21.1q21.2 resulting in a blended phenotype of both Spastic paraplegia 4 and 1q21.1 microduplication syndrome. We believe that the extreme phenotype observed is likely caused by the presence of cells which contain only mutant SPAST, but that the viability of the patient is possible due mosaicism of mutant alleles observed in different proportions across tissues.

ADAM12-directed ectodomain shedding of E-cadherin potentiates trophoblast fusion.

Trophoblasts, placental cells of epithelial lineage, undergo extensive differentiation to form the cellular components of the placenta. Trophoblast progenitor cell differentiation into the multinucleated syncytiotrophoblast is a key developmental process required for placental function, where defects in syncytiotrophoblast formation and turnover associate with placental pathologies and link to poor pregnancy outcomes. The cellular and molecular processes governing syncytiotrophoblast formation are poorly understood, but require the activation of pathways that direct cell fusion. The protease, A Disintegrin and Metalloproteinase 12 (ADAM12), controls cell fusion in myoblasts and is highly expressed in the placenta localizing to multiple trophoblast populations. However, the importance of ADAM12 in regulating trophoblast fusion is unknown. Here, we describe a function for ADAM12 in regulating trophoblast fusion. Using two distinct trophoblast models of cell fusion, we show that ADAM12 is dynamically upregulated and is under the transcriptional control of protein kinase A. siRNA-directed loss of ADAM12 impedes spontaneous fusion of primary cytotrophoblasts, whereas overexpression of the secreted variant, ADAM12S, potentiates cell fusion in the Bewo trophoblast cell line. Mechanistically, both ectopic and endogenous levels of ADAM12 were shown to control trophoblast fusion through E-cadherin ectodomain shedding and remodeling of intercellular boundaries. This study describes a novel role for ADAM12 in placental development, specifically highlighting its importance in controlling the differentiation of villous cytotrophoblasts into multinucleated cellular structures. Moreover, this work identifies E-cadherin as a novel ADAM12 substrate, and highlights the significance that cell adhesion molecule ectodomain shedding has in normal development.

Recurrent triploidy due to a failure to complete maternal meiosis II: whole-exome sequencing reveals candidate variants.

Triploidy is a relatively common cause of miscarriage; however, recurrent triploidy has rarely been reported. A healthy 34-year-old woman was ascertained because of 18 consecutive miscarriages with triploidy found in all 5 karyotyped losses. Molecular results in a sixth loss were also consistent with triploidy. Genotyping of markers near the centromere on multiple chromosomes suggested that all six triploid conceptuses occurred as a result of failure to complete meiosis II (MII). The proband's mother had also experienced recurrent miscarriage, with a total of 18 miscarriages. Based on the hypothesis that an inherited autosomal-dominant maternal predisposition would explain the phenotype, whole-exome sequencing of the proband and her parents was undertaken to identify potential candidate variants. After filtering for quality and rarity, potentially damaging variants shared between the proband and her mother were identified in 47 genes. Variants in genes coding for proteins implicated in oocyte maturation, oocyte activation or polar body extrusion were then prioritized. Eight of the most promising candidate variants were confirmed by Sanger sequencing. These included a novel change in the PLCD4 gene, and a rare variant in the OSBPL5 gene, which have been implicated in oocyte activation upon fertilization and completion of MII. Several variants in genes coding proteins playing a role in oocyte maturation and early embryonic development were also identified. The genes identified may be candidates for the study in other women experiencing recurrent triploidy or recurrent IVF failure.

Activation of endocrine-related gene expression in placental choriocarcinoma cell lines following DNA methylation knock-down.

Increasingly, placental DNA methylation is assessed as a factor in pregnancy-related complications, yet the transcriptional impact of such findings is not always clear. Using a proliferative in vitro placental model, the effect of DNA methylation loss on gene activation was evaluated at a number of genes selected for being differentially methylated in pre-eclampsia-associated placentae in vivo. We aimed to determine whether reduced DNA methylation at specific loci was associated with transcriptional changes at the corresponding gene, thus providing mechanistic underpinnings for previous clinical findings and to assess the degree of transcriptional response amongst our candidate genes. BeWo and JEG3 choriocarcinoma cells were exposed to 1 µM 5-Aza-2'-deoxycytidine (5-Aza-CdR) or vehicle control for 48 h, and re-plated and cultured for a further 72 h in normal media before cells were harvested for RNA and DNA. Bisulphite pyrosequencing confirmed that DNA methylation was reduced by ∼30-50% points at the selected loci studied in both cell lines. Gene activation, measured by qRT-PCR, was highly variable and transcript specific, indicating differential sensitivity to DNA methylation. Most notably, loss of DNA methylation at the leptin (LEP) promoter corresponded to a 200-fold and 40-fold increase in LEP expression in BeWo and JEG3 cells, respectively (P < 0.01). Transcripts of steroidogenic pathway enzymes CYP11A1 and HSD3B1 were up-regulated ∼40-fold in response to 5-Aza-CdR exposure in BeWo cells (P < 0.01). Other transcripts, including aromatase (CYP19), HSD11B2, inhibin (INHBA) and glucocorticoid receptor (NR3C1) were more moderately, although significantly, affected by loss of associated DNA methylation. These data present a mixed effect of DNA methylation changes at selected loci supporting cautionary interpretation of DNA methylation results in the absence of functional data.

Overlapping DNA methylation profile between placentas with trisomy 16 and early-onset preeclampsia.

INTRODUCTION: Maternal preeclampsia is associated with altered placental development in the first trimester of pregnancy. Confined placental trisomy 16 mosaicism (CPM16) is a genetic abnormality of the placenta that is highly predisposing to preeclampsia. We previously demonstrated widespread alterations in DNA methylation in 3rd trimester placentae associated with chromosomally normal early-onset preeclampsia (EOPET) and questioned whether similar changes would be associated with CPM16, making this condition a potential model for studying EOPET-associated changes early in pregnancy. METHODS: Using the Illumina Infinium HumanMethylation450 BeadChip, 3rd trimester CPM16 placental samples (N = 10) were compared to gestational age matched controls, and to 1st trimester trisomy 16 placentae (N = 5). RESULTS: DNA methylation differences associated with CPM16 were identified at 2254 CpGs using stringent criteria (FDR < 0.01, Δß > 0.15). A subset of these differences (11%; p < 0.0001) overlapped those observed in chromosomally normal EOPET using similarly stringent criteria (FDR < 0.01; Δß > 0.125). Importantly, the majority of EOPET-associated CpGs were significantly altered (p < 0.05) in CPM16 with a similar Δß distribution. This was true for CPM16 with (N = 5) and without (N = 5) EOPET, although EOPET cases showed a tendency towards larger changes. Of the shared CPM16/EOPET associated changes, three CpGs near two genes (ARGHEF37 and JUNB) were also altered in 1st trimester trisomy 16 placentae. DISCUSSION: Despite the limited sample size, widespread DNA methylation changes are observed in Trisomy 16 that overlap those seen previously in chromosomally normal EOPET. Hence, Trisomy 16 may provide a model to study the progression of placental changes that occurs in EOPET across different gestational ages.

Beckwith-Wiedemann and Silver-Russell syndromes: opposite developmental imbalances in imprinted regulators of placental function and embryonic growth.

Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are two congenital disorders with opposite outcomes on fetal growth, overgrowth and growth restriction, respectively. Although both disorders are heterogeneous, most cases of BWS and SRS are associated with opposite epigenetic or genetic abnormalities on 11p15.5 leading to opposite imbalances in the expression levels of imprinted genes. In this article, we review evidence implicating these genes in the developmental regulation of embryonic growth and placental function in mouse models. The emerging picture suggests that both SRS and BWS can be caused by the simultaneous and opposite deregulation of two groups of imprinted genes on 11p15.5. A detailed description of the phenotypic abnormalities associated with each syndrome must take into consideration the developmental functions of each gene involved.

Maternal NLRP7 and C6orf221 variants are not a common risk factor for androgenetic moles, triploidy and recurrent miscarriage.

Maternal effect genes control early events of embryogenesis. Maternal homozygous and compound mutations in two such genes, NLRP7 and c6orf221, have been detected in the majority of women experiencing recurrent biparental hydatidiform moles. It was suggested that other forms of reproductive wastage, including diploid androgenetic moles, partial moles, polyploidy, recurrent spontaneous abortions and stillbirths of uncertain etiology, may be caused by NLRP7 or c6orf221 mutations in the mother. To elucidate which subpopulations of women with adverse reproductive outcomes should be screened for NLRP7/C6orf221 variants, we sequenced coding sequence and exon/intron boundaries of NLRP7 and C6orf221 in a well-defined group of 17 women with recurrent miscarriage and additional triploidy or complete hydatidiform moles. The major findings for this group were non-synonymous variants of NLRP7, rather than clearly pathogenic mutations. To assess the role of these variants, we genotyped them in a larger group including women with primary recurrent miscarriage (n = 39), paternal triploid conceptions (n = 22) and women with proven fertility after age 37 and no prior history of miscarriage or pregnancy complications (n = 52). No associations between non-synonymous NLRP7 variants and primary recurrent miscarriage or partial hydatidiform molar pregnancies were detected. Our findings suggest that neither mutations nor variants in NLRP7 and C6orf221 are major factors contributing to the risk of these types of pregnancy complications. Further studies in larger groups of patients and controls are needed to specify the impact of NLRP7 rare non-synonymous variants on genetic susceptibility to recurrent reproductive wastage.

Prenatal and perinatal environmental influences on the human fetal and placental epigenome.

Drug, hormonal, and nutritional exposures in pregnancy can cause birth defects, or have more subtle influences that may affect adult health through epigenetic developmental programming. Characterizing these epigenetic changes may provide valuable insight into the mechanism by which our health is influenced by the environment and provide biomarkers of exposure; however, currently there are few data from human studies directly addressing this. We review data on three types of exposures-nutritional, glucocorticoid, and endocrine-disrupting chemicals-and discuss how these may be associated with epigenetic changes in the fetus and the placenta, a key mediator of in utero environment. In addition, we outline some considerations for investigation of epigenetic effects in the fetus and placenta that are important to consider in the design of studies for the future.

Patterns of placental development evaluated by X chromosome inactivation profiling provide a basis to evaluate the origin of epigenetic variation.

BACKGROUND: Inactivation of the maternally or paternally derived X chromosome (XCI) initially occurs in a random manner in early development; however as tissues form, a 'patchiness' will occur in terms of which X is inactivated if cells positioned near each other are clonally descended from a common precursor. Determining the relationship between skewed XCI in different tissues and in different samples from the same tissue provides a molecular assessment of the developmental history of a particular tissue that can then be used to understand how genetic and epigenetic variation arises in development. METHODS: XCI skewing was evaluated in and compared between amnion, chorion, trophoblast and mesenchyme using multiple sampling sites from 14 term placentae. XCI was also evaluated in chorionic villus samples obtained at multiple sites and depths from four additional term placentae. The pattern of variation was then compared with methylation variation associated with the H19/IGF2 imprinting control region (ICR); promoter regions of KISS1, PTPN6, CASP8 and APC; and LINE-1 elements. RESULTS: Mean placental level of skewing for amnion and chorion are correlated, consistent with a common developmental origin of at least a component of these membranes from inner cell mass derivatives subsequent to XCI, while trophoblast appears to be derived independently, consistent with its origin from the trophectoderm. Villus samples taken from different depths spanning the fetal to maternal side of the placenta were highly clonally related. Comparing patterns of clonal growth identified through XCI to the distribution of epigenetic variation in other genomic regions suggests that some variation arises early in development (e.g. LINE-1 methylation), whereas other variation arises predominantly after villus tree formation (e.g. methylation at H19/IGF2 ICR). CONCLUSIONS: The patterns of XCI skewing are consistent with a model whereby each biopsied site of chorionic villi represents one or a few individual villus trees, each of which is clonally derived from only one or a few precursor cells. Sampling of placentae to evaluate changes associated with clinical pathology should be done with consideration of the tree-to-tree differences. A limitation of this study is the small number of placentas used and therefore placental-specific differences in variation could not be assessed.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

Review: A high capacity of the human placenta for genetic and epigenetic variation: implications for assessing pregnancy outcome.

Genetic and epigenetic studies of the human placenta can help to clarify the underlying mechanisms of placenta-associated diseases. However, such studies have also revealed a considerable degree of within- and between-placenta variability, which can be attributed to a variety of influences. We illustrate the inherent heterogeneity in the placenta using examples from two types of studies: 1) chromosomal mosaicism and 2) DNA methylation variation. We discuss the factors that may influence the distribution of variation and how, understanding the source of this variation is important for interpreting data used to investigate and predict clinical outcomes.

The utility of quantitative methylation assays at imprinted genes for the diagnosis of fetal and placental disorders.

An imbalance of imprinted gene expression within 11p15.5 is observed in Beckwith-Wiedemann syndrome (BWS), as well as in a variety of placental abnormalities including complete hydatidiform mole (CHM), placental mesenchymal dysplasia (PMD) and triploidy. To facilitate the diagnosis of epigenetic errors and chromosomal imbalance of 11p15.5, we validated a pyrosequencing assay to measure methylation at KvDMR1 using blood samples from 13 BWS cases, 8 of which showed reduced methylation as compared to control blood. An imbalance between maternal and paternal genomes as is found in triploidy, CHM or PMD was also associated with altered KvDMR1 methylation. A reciprocal pattern of methylation was obtained in the triploid cases by assaying the proximal 11p15.5 ICR associated with H19. To distinguish chromosome 11 specific alterations from whole genome imbalance, other imprinted differentially methylated regions (DMRs) can be utilized. Thus, pyrosequencing assays for DMRs associated with SGCE, SNRPN, and MEST were also compared for their utility in diagnosing parental imbalance in placental samples. While each of these assays could successfully distinguish parental origin of triploidy, SGCE showed the clearest separation between groups. The combined use of a chromosome 11p15.5 assay (e.g. KvDMR1 or H19-ICR) and non-chromosome 11 assay (e.g. SGCE) provides a potentially valuable diagnostic tool in the rapid screening of methylation errors in placental disorders. These results also show the maintenance of imprinting status at these loci in the human placenta, even in the presence of abnormal pathology.

Evaluating DNA methylation and gene expression variability in the human term placenta.

UNLABELLED: Obtaining representative samples from a term placenta for gene-expression studies is confounded by both within placental heterogeneity and sampling effects such as sample location and processing time. Epigenetic processes involved in the regulation of gene expression, such as DNA methylation, may show similar variability, but are less well studied. Therefore, we investigated the nature of within and between- placenta variation in gene expression and DNA methylation of genes that were chosen for being differentially expressed or methylated by cell type within the placenta. METHODS: In total, two or more samples from each of 38 normal term placentae were utilized. The expression levels of CDH1, CDH11, ID2, PLAC1 and KISS1 were evaluated by real-time PCR. DNA methylation levels of LINE1 elements and CpGs within the promoter regions of KISS1, PTPN6, CASP8, and APC were similarly quantified by pyrosequencing. RESULTS: Despite considerable sample-to-sample variability within each placenta, the within-placenta correlation for both gene expression and methylation was significant for each studied gene. Most of this variability was not due to sample location. However, between placental differences in gene expression were inflated by the dramatic effect of processing time (0-24 h) on mRNA levels, particularly for PLAC1 and KISS1 (both expressed in the apical syncytiotrophoblast). In contrast, DNA methylation levels remained relatively constant over this same time period. CONCLUSION: Due to extensive site-to-site variability, multiple sampled sites are needed to accurately represent a placenta for molecular studies. Furthermore, mRNA quantitation of some genes may be hampered by its rapid degradation post-delivery (and possibly perinatally) and thus processing time should be considered in such analyses. Within-placenta correlations in expression and methylation from unrelated genes raise the possibility that methylation and expression variation may potentially reflect cell composition differences between samples rather than true differences occurring at the cellular level.

Identification of copy number variants in miscarriages from couples with idiopathic recurrent pregnancy loss.

BACKGROUND: Recurrent pregnancy loss (RPL), defined as two or more miscarriages, affects 3-5% of couples trying to establish a family. Despite extensive evaluation, no factor is identified in ∼40% of cases. In this study, we investigated the possibility that submicroscopic chromosomal changes, not detectable by conventional cytogenetic analysis, exist in miscarriages with normal karyotypes (46,XY or 46,XX) from couples with idiopathic RPL. METHODS: Array comparative genomic hybridization (array-CGH) was used to assess for DNA copy number variants (CNVs) in 26 miscarriages with normal karyotypes. Parental array-CGH analysis was performed to determine if miscarriage CNVs were de novo or inherited. RESULTS: There were 11 unique (previously not described) CNVs, all inherited, identified in 13 miscarriages from 8 couples. The maternal origin of two CNVs was of interest as they involved the imprinted genes TIMP2 and CTNNA3, which are only normally expressed from the maternal copy in the placenta. Two additional cohorts, consisting of 282 women with recurrent miscarriage (RM) and 61 fertile women, were screened for these two CNVs using a Quantitative Multiplex Fluorescent PCR of Short Fragments assay. One woman with RM, but none of the fertile women, carried the CTNNA3-associated CNV. CONCLUSIONS: This preliminary study shows that array-CGH is useful for detecting CNVs in cases of RPL. Further investigations of CNVs, particularly those involving genes that are imprinted in placenta, in women with RPL could be worthwhile.

Decreased placental methylation at the H19/IGF2 imprinting control region is associated with normotensive intrauterine growth restriction but not preeclampsia.

UNLABELLED: Many genes exhibiting genomic imprinting, parent-of-origin differences in gene expression, are involved in regulating placental and fetal growth. The goal of the present study was to assess whether abnormal regulation of imprinted genes is associated with intrauterine growth restriction (IUGR) and/or preeclampsia (PET). METHODS: Genomic DNA was extracted from at least two whole villi samples from control (N=22), IUGR (N=13), PET (N=17), and PET+IUGR (N=21) placentas. Methylation was assessed using the Illumina GoldenGate Methylation Cancer Panel I array and Pyrosequencing and MS-SNuPE assays. RESULTS: The 11p15.5 ICR1 (associated with H19 and IGF2) methylation showed considerable intra-placental variability. Nonetheless, average methylation at this site was significantly decreased in normotensive IUGR placentas (p<0.001), but not in any other group. Methylation at ICR2 (KvDMR1; associated with CDKN1C and other maternally expressed 11p15.5 genes) was not significantly altered in any group and no significant changes in expression levels were observed in the genes controlled by this region. There were no significant methylation changes observed in any candidate imprinted gene evaluated by the Illumina array. LINE-1 methylation, a marker of whole genome methylation, was also similar in all groups. CONCLUSIONS: Reduced methylation of ICR1 is associated with normotensive IUGR but not IUGR associated with preeclampsia, suggesting a different etiology of IUGR in this group. A reduction in placental IGF2 could be an adaptive response to restrict fetal growth in the presence of abnormal placentation or a response to poor fetal growth itself.

Placental mesenchymal dysplasia associated with fetal overgrowth and mosaic deletion of the maternal copy of 11p15.5.

Placentae with mesenchymal dysplasia (PMD) are typically larger than average and show cystic areas on ultrasonography. Fetal outcomes are variable and are often associated with growth restriction. However, enigmatically, some associated fetuses show signs of Beckwith-Wiedemann syndrome (BWS). PMD has recently been shown to result from androgenetic (complete paternal uniparental disomy) chimerism in the placenta in pregnancies that were associated with some fetal growth restriction. Cases of PMD associated with overgrowth have not previously been investigated molecularly. We present a case of focal PMD associated with a male fetus showing overgrowth with an enlarged heart, marked fetal ascites and intrauterine fetal death at 34 weeks, but no other BWS manifestations. Mosaicism for an unbalanced translocation leading to deletion of the maternal copy of the BWS region on 11p15.5 and partial duplication of 17q was observed in placenta, but not fetal samples. While the placental findings of PMD can be caused by an unbalanced dosage of genes in 11p15.5 alone, fetal growth parameters appear to depend on the underlying mechanism and likely also the level and distribution of abnormal cells.

Toll-like receptor 4 polymorphisms and idiopathic chromosomally normal miscarriage.

BACKGROUND: Lipopolysaccharide (LPS or endotoxin) exposure resulting from microbial invasion of the endometrium disturbs the Th1/Th2 balance at the feto-maternal interface and has been linked to the risk of idiopathic miscarriage in a range of human and animal studies. Toll-like receptor 4 (TLR4) mediates LPS signalling, and the human TLR4 gene harbours two single-nucleotide polymorphisms (SNPs) known to reduce LPS responsiveness. We hypothesized that genetic variation altering TLR4 function may influence the risk of idiopathic pregnancy loss. METHODS AND RESULTS: We examined fetal TLR4 genotypes in a case-control cohort of chromosomally normal miscarriages (n=96) and healthy term newborns (n=113). The allele frequencies of the Asp299Gly and Thr399Ile TLR4 SNPs were determined by quantitative PCR using DNA extracted from extraembryonic tissues and umbilical cord blood, respectively. TLR4 genotype frequencies were not significantly different between cases and controls. CONCLUSIONS: There was no association between fetal TLR4 polymorphisms, Asp299Gly and Thr399Ile, known to blunt LPS responsiveness, and the risk of idiopathic, chromosomally normal miscarriage. Nevertheless, TLR4 or perhaps other LPS-binding chaperone molecules are biologically plausible candidate genes that may alter the risk of idiopathic miscarriage.

Pregnancy and postnatal outcome of mosaic isochromosome 20q.

Prenatally diagnosed mosaicism for isochromosome 20q is generally reported in association with a normal outcome at birth and is rarely confirmed postnatally. However, the origin of these abnormal cells is unclear and there are few reports of long-term outcomes. We present an additional case of prenatally detected isochromosome 20q, with normal outcome up to age 3.6 years. The abnormal cells, while present at high levels in the amniotic fluid, could not be confirmed in placenta or fetal blood. Nonetheless, based on a review of the literature, the level of isochromosome 20q cells found is associated with risk of abnormal outcome, suggesting a possible effect in some cases.