The human placenta exhibits a unique transcriptomic void.

The human placenta exhibits a unique genomic architecture with an unexpectedly high mutation burden and many uniquely expressed genes. The aim of this study is to identify transcripts that are uniquely absent or depleted in the placenta. Here, we show that 40 of 46 of the other organs have no selectively depleted transcripts and that, of the remaining six, the liver has the largest number, with 26. In contrast, the term placenta has 762 depleted transcripts. Gene Ontology analysis of this depleted set highlighted multiple pathways reflecting known unique elements of placental physiology. For example, transcripts associated with neuronal function are in the depleted set-as expected given the lack of placental innervation. However, this demonstrated overrepresentation of genes involved in mitochondrial function (p = 5.8 × 10-10), including PGC-1α, the master regulator of mitochondrial biogenesis, and genes involved in polyamine metabolism (p = 2.1 × 10-4).

Increased Placental sFLT1 (Soluble fms-Like Tyrosine Kinase Receptor-1) Drives the Antiangiogenic Profile of Maternal Serum Preceding Preeclampsia but Not Fetal Growth Restriction.

BACKGROUND: Preeclampsia and fetal growth restriction (FGR) are both associated with an increased ratio of sFLT1 (soluble fms-like tyrosine kinase-1) to PlGF (placenta growth factor) in maternal serum. In preeclampsia, it is assumed that increased placental release of sFLT1 results in PlGF being bound and inactivated. However, direct evidence for this model is incomplete, and it is unclear whether the same applies in FGR. METHODS: We conducted a prospective cohort study where we followed 4212 women having first pregnancies from their dating ultrasound, obtained blood samples serially through the pregnancy, and performed systematic sampling of the placenta after delivery. The aim of the present study was to determine the relationship between protein levels of sFLT1 and PlGF in maternal serum measured at ≈36 weeks and placental tissue lysates obtained after term delivery in 82 women with preeclampsia, 50 women with FGR, and 132 controls. RESULTS: The sFLT1:PlGF ratio was increased in both preeclampsia and FGR in both the placenta and maternal serum. However, in preeclampsia, the maternal serum ratio of sFLT1:PlGF was strongly associated with placental sFLT1 level (r=0.45; P<0.0001) but not placental PlGF level (r=-0.17; P=0.16). In contrast, in FGR, the maternal serum ratio of sFLT1:PlGF was strongly associated with placental PlGF level (r=-0.35; P=0.02) but not sFLT1 level (r=0.04; P=0.81). CONCLUSIONS: We conclude that the elevated sFLT1:PlGF ratio is primarily driven by increased placental sFLT1 in preeclampsia, whereas in FGR, it is primarily driven by decreased placental PlGF.

Placental sex-dependent spermine synthesis regulates trophoblast gene expression through acetyl-coA metabolism and histone acetylation.

Placental function and dysfunction differ by sex but the mechanisms are unknown. Here we show that sex differences in polyamine metabolism are associated with escape from X chromosome inactivation of the gene encoding spermine synthase (SMS). Female placental trophoblasts demonstrate biallelic SMS expression, associated with increased SMS mRNA and enzyme activity. Polyamine depletion in primary trophoblasts reduced glycolysis and oxidative phosphorylation resulting in decreased acetyl-coA availability and global histone hypoacetylation in a sex-dependent manner. Chromatin-immunoprecipitation sequencing and RNA-sequencing identifies progesterone biosynthesis as a target of polyamine regulated gene expression, and polyamine depletion reduced progesterone release in male trophoblasts. The effects of polyamine depletion can be attributed to spermine as SMS-silencing recapitulated the effects on energy metabolism, histone acetylation, and progesterone release. In summary, spermine metabolism alters trophoblast gene expression through acetyl-coA biosynthesis and histone acetylation, and SMS escape from X inactivation explains some features of human placental sex differences.

The RNA landscape of the human placenta in health and disease.

The placenta is the interface between mother and fetus and inadequate function contributes to short and long-term ill-health. The placenta is absent from most large-scale RNA-Seq datasets. We therefore analyze long and small RNAs (~101 and 20 million reads per sample respectively) from 302 human placentas, including 94 cases of preeclampsia (PE) and 56 cases of fetal growth restriction (FGR). The placental transcriptome has the seventh lowest complexity of 50 human tissues: 271 genes account for 50% of all reads. We identify multiple circular RNAs and validate 6 of these by Sanger sequencing across the back-splice junction. Using large-scale mass spectrometry datasets, we find strong evidence of peptides produced by translation of two circular RNAs. We also identify novel piRNAs which are clustered on Chr1 and Chr14. PE and FGR are associated with multiple and overlapping differences in mRNA, lincRNA and circRNA but fewer consistent differences in small RNAs. Of the three protein coding genes differentially expressed in both PE and FGR, one encodes a secreted protein FSTL3 (follistatin-like 3). Elevated serum levels of FSTL3 in pregnant women are predictive of subsequent PE and FGR. To aid visualization of our placenta transcriptome data, we develop a web application ( https://www.obgyn.cam.ac.uk/placentome/ ).

Fetal inheritance of chromosomally integrated human herpesvirus 6 predisposes the mother to pre-eclampsia.

Pre-eclampsia (typically characterized by new-onset hypertension and proteinuria in the second half of pregnancy) represents a major determinant of the global burden of disease1,2. Its pathophysiology involves placental dysfunction, but the mechanism is unclear. Viral infection can cause organ dysfunction, but its role in placentally related disorders of human pregnancy is unknown3. We addressed this using RNA sequencing metagenomics4-6 of placental samples from normal and complicated pregnancies. Here, we show that human herpesvirus 6 (HHV-6, A or B) RNA was detected in 6.1% of cases of pre-eclampsia and 2.2% of other pregnancies. Fetal genotyping demonstrated that 70% of samples with HHV-6 RNA in the placenta exhibited inherited, chromosomally integrated HHV-6 (iciHHV-6). We genotyped 467 pre-eclampsia cases and 3,854 controls and found an excess of iciHHV-6 in the cases (odds ratio of 2.8, 95% confidence intervals of 1.4-5.6, P = 0.008). We validated this finding by comparing iciHHV-6 in a further 740 cases with controls from large-scale population studies (odds ratio of 2.5, 95% confidence intervals of 1.4-4.4, P = 0.0013). We conclude that iciHHV-6 results in the transcription of viral RNA in the human placenta and predisposes the mother to pre-eclampsia.

Placental polyamine metabolism differs by fetal sex, fetal growth restriction, and preeclampsia.

Preeclampsia and fetal growth restriction (FGR) are major causes of the more than 5 million perinatal and infant deaths occurring globally each year, and both are associated with placental dysfunction. The risk of perinatal and infant death is greater in males, but the mechanisms are unclear. We studied data and biological samples from the Pregnancy Outcome Prediction (POP) study, a prospective cohort study that followed 4,212 women having first pregnancies from their dating ultrasound scan through delivery. We tested the hypothesis that fetal sex would be associated with altered placental function using multiomic and targeted analyses. We found that spermine synthase (SMS) escapes X-chromosome inactivation (XCI) in the placenta and is expressed at lower levels in male primary trophoblast cells, and male cells were more sensitive to polyamine depletion. The spermine metabolite N1,N12-diacetylspermine (DiAcSpm) was higher in the female placenta and in the serum of women pregnant with a female fetus. Higher maternal serum levels of DiAcSpm increased the risk of preeclampsia but decreased the risk of FGR. To our knowledge, DiAcSpm is the first maternal biomarker to demonstrate opposite associations with preeclampsia and FGR, and this is the first evidence to implicate polyamine metabolism in sex-related differences in placentally related complications of human pregnancy.

ELABELA/APELA Levels Are Not Decreased in the Maternal Circulation or Placenta among Women with Preeclampsia.

The genetic deletion of apelin receptor early endogenous ligand (Elabela; official name APELA) produces a preeclampsia-like phenotype in mice. However, evidence linking ELABELA with human disease is lacking. Therefore, we measured placental mRNA and circulating ELABELA in human samples. ELABELA mRNA (measured by RNA sequencing) was unchanged in 82 preeclamptic placentas compared with 82 matched controls (mean difference, 0.53%; 95% CI, -25.9 to 27.0; P = 0.78). We measured circulating ELABELA in 32 women with preterm preeclampsia (delivered at <34 weeks' gestation) and 32 matched controls sampled at the same gestational age. There was no difference in circulating ELABELA concentrations in the preeclamptic cohort compared with controls (median, 28.5 pg/mL; 95% CI, 5.3 to 63.2 versus median, 20.5 pg/mL; 95% CI, 9.2 to 58.0, respectively); the median difference was 8.0 pg/mL (95% CI, -17.7 to 12.1; P = 0.43). In contrast, soluble FLT1 (a protein with an established association with preeclampsia) mRNA was increased in placental tissue (mean difference, 34.9%; 95% CI, 16.6 to 53.1; P = 0.001), and circulating concentrations were 16.8-fold higher among the preeclamptic cohort (P < 0.0001). In conclusion, we were able to recapitulate the association between circulating soluble FLT1 and preeclampsia, but there was no association with ELABELA. The speculated clinical relevance of observations in the murine model linking ELABELA to preeclampsia likely are incorrect.

Genome-wide oxidative bisulfite sequencing identifies sex-specific methylation differences in the human placenta.

DNA methylation is an important regulator of gene function. Fetal sex is associated with the risk of several specific pregnancy complications related to placental function. However, the association between fetal sex and placental DNA methylation remains poorly understood. We carried out whole-genome oxidative bisulfite sequencing in the placentas of two healthy female and two healthy male pregnancies generating an average genome depth of coverage of 25x. Most highly ranked differentially methylated regions (DMRs) were located on the X chromosome but we identified a 225 kb sex-specific DMR in the body of the CUB and Sushi Multiple Domains 1 (CSMD1) gene on chromosome 8. The sex-specific differential methylation pattern observed in this region was validated in additional placentas using in-solution target capture. In a new RNA-seq data set from 64 female and 67 male placentas, CSMD1 mRNA was 1.8-fold higher in male than in female placentas (P value = 8.5 × 10-7, Mann-Whitney test). Exon-level quantification of CSMD1 mRNA from these 131 placentas suggested a likely placenta-specific CSMD1 isoform not detected in the 21 somatic tissues analyzed. We show that the gene body of an autosomal gene, CSMD1, is differentially methylated in a sex- and placental-specific manner, displaying sex-specific differences in placental transcript abundance.

RNA-seq reveals conservation of function among the yolk sacs of human, mouse, and chicken.

The yolk sac is phylogenetically the oldest of the extraembryonic membranes. The human embryo retains a yolk sac, which goes through primary and secondary phases of development, but its importance is controversial. Although it is known to synthesize proteins, its transport functions are widely considered vestigial. Here, we report RNA-sequencing (RNA-seq) data for the human and murine yolk sacs and compare those data with data for the chicken. We also relate the human RNA-seq data to proteomic data for the coelomic fluid bathing the yolk sac. Conservation of transcriptomes across the species indicates that the human secondary yolk sac likely performs key functions early in development, particularly uptake and processing of macro- and micronutrients, many of which are found in coelomic fluid. More generally, our findings shed light on evolutionary mechanisms that give rise to complex structures such as the placenta. We identify genetic modules that are conserved across mammals and birds, suggesting these modules are part of the core amniote genetic repertoire and are the building blocks for both oviparous and viviparous reproductive modes. We propose that although a choriovitelline placenta is never established physically in the human, the placental villi, the exocoelomic cavity, and the secondary yolk sac function together as a physiological equivalent.

MANORAA (Mapping Analogous Nuclei Onto Residue And Affinity) for identifying protein-ligand fragment interaction, pathways and SNPs.

Protein-ligand interaction analysis is an important step of drug design and protein engineering in order to predict the binding affinity and selectivity between ligands to the target proteins. To date, there are more than 100 000 structures available in the Protein Data Bank (PDB), of which ∼30% are protein-ligand (MW below 1000 Da) complexes. We have developed the integrative web server MANORAA (Mapping Analogous Nuclei Onto Residue And Affinity) with the aim of providing a user-friendly web interface to assist structural study and design of protein-ligand interactions. In brief, the server allows the users to input the chemical fragments and present all the unique molecular interactions to the target proteins with available three-dimensional structures in the PDB. The users can also link the ligands of interest to assess possible off-target proteins, human variants and pathway information using our all-in-one integrated tools. Taken together, we envisage that the server will facilitate and improve the study of protein-ligand interactions by allowing observation and comparison of ligand interactions with multiple proteins at the same time. (http://manoraa.org).

Integrated allelic, transcriptional, and phenomic dissection of the cardiac effects of titin truncations in health and disease.

The recent discovery of heterozygous human mutations that truncate full-length titin (TTN, an abundant structural, sensory, and signaling filament in muscle) as a common cause of end-stage dilated cardiomyopathy (DCM) promises new prospects for improving heart failure management. However, realization of this opportunity has been hindered by the burden of TTN-truncating variants (TTNtv) in the general population and uncertainty about their consequences in health or disease. To elucidate the effects of TTNtv, we coupled TTN gene sequencing with cardiac phenotyping in 5267 individuals across the spectrum of cardiac physiology and integrated these data with RNA and protein analyses of human heart tissues. We report diversity of TTN isoform expression in the heart, define the relative inclusion of TTN exons in different isoforms (using the TTN transcript annotations available at http://cardiodb.org/titin), and demonstrate that these data, coupled with the position of the TTNtv, provide a robust strategy to discriminate pathogenic from benign TTNtv. We show that TTNtv is the most common genetic cause of DCM in ambulant patients in the community, identify clinically important manifestations of TTNtv-positive DCM, and define the penetrance and outcomes of TTNtv in the general population. By integrating genetic, transcriptome, and protein analyses, we provide evidence for a length-dependent mechanism of disease. These data inform diagnostic criteria and management strategies for TTNtv-positive DCM patients and for TTNtv that are identified as incidental findings.

NECTAR: a database of codon-centric missense variant annotations.

NECTAR (Non-synonymous Enriched Coding muTation ARchive; http://nectarmutation.org) is a database and web application to annotate disease-related and functionally important amino acids in human proteins. A number of tools are available to facilitate the interpretation of DNA variants identified in diagnostic or research sequencing. These typically identify previous reports of DNA variation at a given genomic location, predict its effects on transcript and protein sequence and may predict downstream functional consequences. Previous reports and functional annotations are typically linked by the genomic location of the variant observed. NECTAR collates disease-causing variants and functionally important amino acid residues from a number of sources. Importantly, rather than simply linking annotations by a shared genomic location, NECTAR annotates variants of interest with details of previously reported variation affecting the same codon. This provides a much richer data set for the interpretation of a novel DNA variant. NECTAR also identifies functionally equivalent amino acid residues in evolutionarily related proteins (paralogues) and, where appropriate, transfers annotations between them. As well as accessing these data through a web interface, users can upload batches of variants in variant call format (VCF) for annotation on-the-fly. The database is freely available to download from the ftp site: ftp://ftp.nectarmutation.org.

Next generation diagnostics in inherited arrhythmia syndromes : a comparison of two approaches.

Next-generation sequencing (NGS) provides an unprecedented opportunity to assess genetic variation underlying human disease. Here, we compared two NGS approaches for diagnostic sequencing in inherited arrhythmia syndromes. We compared PCR-based target enrichment and long-read sequencing (PCR-LR) with in-solution hybridization-based enrichment and short-read sequencing (Hyb-SR). The PCR-LR assay comprehensively assessed five long-QT genes routinely sequenced in diagnostic laboratories and "hot spots" in RYR2. The Hyb-SR assay targeted 49 genes, including those in the PCR-LR assay. The sensitivity for detection of control variants did not differ between approaches. In both assays, the major limitation was upstream target capture, particular in regions of extreme GC content. These initial experiences with NGS cardiovascular diagnostics achieved up to 89 % sensitivity at a fraction of current costs. In the next iteration of these assays we anticipate sensitivity above 97 % for all LQT genes. NGS assays will soon replace conventional sequencing for LQT diagnostics and molecular pathology.

MetaBase--the wiki-database of biological databases.

Biology is generating more data than ever. As a result, there is an ever increasing number of publicly available databases that analyse, integrate and summarize the available data, providing an invaluable resource for the biological community. As this trend continues, there is a pressing need to organize, catalogue and rate these resources, so that the information they contain can be most effectively exploited. MetaBase (MB) (http://MetaDatabase.Org) is a community-curated database containing more than 2000 commonly used biological databases. Each entry is structured using templates and can carry various user comments and annotations. Entries can be searched, listed, browsed or queried. The database was created using the same MediaWiki technology that powers Wikipedia, allowing users to contribute on many different levels. The initial release of MB was derived from the content of the 2007 Nucleic Acids Research (NAR) Database Issue. Since then, approximately 100 databases have been manually collected from the literature, and users have added information for over 240 databases. MB is synchronized annually with the static Molecular Biology Database Collection provided by NAR. To date, there have been 19 significant contributors to the project; each one is listed as an author here to highlight the community aspect of the project.

Meet me halfway: when genomics meets structural bioinformatics.

The DNA sequencing technology developed by Frederick Sanger in the 1970s established genomics as the basis of comparative genetics. The recent invention of next-generation sequencing (NGS) platform has added a new dimension to genome research by generating ultra-fast and high-throughput sequencing data in an unprecedented manner. The advent of NGS technology also provides the opportunity to study genetic diseases where sequence variants or mutations are sought to establish a causal relationship with disease phenotypes. However, it is not a trivial task to seek genetic variants responsible for genetic diseases and even harder for complex diseases such as diabetes and cancers. In such polygenic diseases, multiple genes and alleles, which can exist in healthy individuals, come together to contribute to common disease phenotypes in a complex manner. Hence, it is desirable to have an approach that integrates omics data with both knowledge of protein structure and function and an understanding of networks/pathways, i.e. functional genomics and systems biology; in this way, genotype-phenotype relationships can be better understood. In this review, we bring this 'bottom-up' approach alongside the current NGS-driven genetic study of genetic variations and disease aetiology. We describe experimental and computational techniques for assessing genetic variants and their deleterious effects on protein structure and function.

Structural and functional restraints on the occurrence of single amino acid variations in human proteins.

Human genetic variation is the incarnation of diverse evolutionary history, which reflects both selectively advantageous and selectively neutral change. In this study, we catalogue structural and functional features of proteins that restrain genetic variation leading to single amino acid substitutions. Our variation dataset is divided into three categories: i) Mendelian disease-related variants, ii) neutral polymorphisms and iii) cancer somatic mutations. We characterize structural environments of the amino acid variants by the following properties: i) side-chain solvent accessibility, ii) main-chain secondary structure, and iii) hydrogen bonds from a side chain to a main chain or other side chains. To address functional restraints, amino acid substitutions in proteins are examined to see whether they are located at functionally important sites involved in protein-protein interactions, protein-ligand interactions or catalytic activity of enzymes. We also measure the likelihood of amino acid substitutions and the degree of residue conservation where variants occur. We show that various types of variants are under different degrees of structural and functional restraints, which affect their occurrence in human proteome.

MitoInteractome: mitochondrial protein interactome database, and its application in 'aging network' analysis.

BACKGROUND: Mitochondria play a vital role in the energy production and apoptotic process of eukaryotic cells. Proteins in the mitochondria are encoded by nuclear and mitochondrial genes. Owing to a large increase in the number of identified mitochondrial protein sequences and completed mitochondrial genomes, it has become necessary to provide a web-based database of mitochondrial protein information. RESULTS: We present 'MitoInteractome', a consolidated web-based portal containing a wealth of information on predicted protein-protein interactions, physico-chemical properties, polymorphism, and diseases related to the mitochondrial proteome. MitoInteractome contains 6,549 protein sequences which were extracted from the following databases: SwissProt, MitoP, MitoProteome, HPRD and Gene Ontology database. The first general mitochondrial interactome has been constructed based on the concept of 'homologous interaction' using PSIMAP (Protein Structural Interactome MAP) and PEIMAP (Protein Experimental Interactome MAP). Using the above mentioned methods, protein-protein interactions were predicted for 74 species. The mitochondrial protein interaction data of humans was used to construct a network for the aging process. Analysis of the 'aging network' gave us vital insights into the interactions among proteins that influence the aging process. CONCLUSION: MitoInteractome is a comprehensive database that would (1) aid in increasing our understanding of the molecular functions and interaction networks of mitochondrial proteins, (2) help in identifying new target proteins for experimental research using predicted protein-protein interaction information, and (3) help in identifying biomarkers for diagnosis and new molecular targets for drug development related to mitochondria. MitoInteractome is available at http://mitointeractome.kobic.kr/.

Structural and functional constraints in the evolution of protein families.

High-throughput genomic sequencing has focused attention on understanding differences between species and between individuals. When this genetic variation affects protein sequences, the rate of amino acid substitution reflects both Darwinian selection for functionally advantageous mutations and selectively neutral evolution operating within the constraints of structure and function. During neutral evolution, whereby mutations accumulate by random drift, amino acid substitutions are constrained by factors such as the formation of intramolecular and intermolecular interactions and the accessibility to water or lipids surrounding the protein. These constraints arise from the need to conserve a specific architecture and to retain interactions that mediate functions in protein families and superfamilies.

Structural interactomics: informatics approaches to aid the interpretation of genetic variation and the development of novel therapeutics.

Here we review the use of informatics in structural interactomics, with particular emphasis on understanding interfacial contacts in the development of novel therapeutics and the interpretation of genetic variation. We describe the availability and applicability of structural databases of protein interactions which facilitate this endeavour. We demonstrate the applicability of a structural interactomics approach to the study of the fibroblast growth factor (FGF)-stimulated mitogen-activated protein kinase (MAPK) pathway.

Structural and functional restraints in the evolution of protein families and superfamilies.

Divergent evolution of proteins reflects both selectively advantageous and neutral amino acid substitutions. In the present article, we examine restraints on sequence, which arise from selectively advantageous roles for structure and function and which lead to the conservation of local sequences and structures in families and superfamilies. We analyse structurally aligned members of protein families and superfamilies in order to investigate the importance of the local structural environment of amino acid residues in the acceptance of amino acid substitutions during protein evolution. We show that solvent accessibility is the most important determinant, followed by the existence of hydrogen bonds from the side-chain to main-chain functions and the nature of the element of secondary structure to which the amino acid contributes. Polar side chains whose hydrogen-bonding potential is satisfied tend to be more conserved than their unsatisfied or non-hydrogen-bonded counterparts, and buried and satisfied polar residues tend to be significantly more conserved than buried hydrophobic residues. Finally, we discuss the importance of functional restraints in the form of interactions of proteins with other macromolecules in assemblies or with substrates, ligands or allosteric regulators. We show that residues involved in such functional interactions are significantly more conserved and have differing amino acid substitution patterns.