Evolutionarily Conserved Principles Predict 3D Chromatin Organization.

Topologically associating domains (TADs), CTCF loop domains, and A/B compartments have been identified as important structural and functional components of 3D chromatin organization, yet the relationship between these features is not well understood. Using high-resolution Hi-C and HiChIP, we show that Drosophila chromatin is organized into domains we term compartmental domains that correspond precisely with A/B compartments at high resolution. We find that transcriptional state is a major predictor of Hi-C contact maps in several eukaryotes tested, including C. elegans and A. thaliana. Architectural proteins insulate compartmental domains by reducing interaction frequencies between neighboring regions in Drosophila, but CTCF loops do not play a distinct role in this organism. In mammals, compartmental domains exist alongside CTCF loop domains to form topological domains. The results suggest that compartmental domains are responsible for domain structure in all eukaryotes, with CTCF playing an important role in domain formation in mammals.

Analysis of Hi-C data using SIP effectively identifies loops in organisms from C. elegans to mammals.

Chromatin loops are a major component of 3D nuclear organization, visually apparent as intense point-to-point interactions in Hi-C maps. Identification of these loops is a critical part of most Hi-C analyses. However, current methods often miss visually evident CTCF loops in Hi-C data sets from mammals, and they completely fail to identify high intensity loops in other organisms. We present SIP, Significant Interaction Peak caller, and SIPMeta, which are platform independent programs to identify and characterize these loops in a time- and memory-efficient manner. We show that SIP is resistant to noise and sequencing depth, and can be used to detect loops that were previously missed in human cells as well as loops in other organisms. SIPMeta corrects for a common visualization artifact by accounting for Manhattan distance to create average plots of Hi-C and HiChIP data. We then demonstrate that the use of SIP and SIPMeta can lead to biological insights by characterizing the contribution of several transcription factors to CTCF loop stability in human cells. We also annotate loops associated with the SMC component of the dosage compensation complex (DCC) in Caenorhabditis elegans and demonstrate that loop anchors represent bidirectional blocks for symmetrical loop extrusion. This is in contrast to the asymmetrical extrusion until unidirectional blockage by CTCF that is presumed to occur in mammals. Using HiChIP and multiway ligation events, we then show that DCC loops form a network of strong interactions that may contribute to X Chromosome-wide condensation in C. elegans hermaphrodites.

Placental microRNAs relate to early childhood growth trajectories.

BACKGROUND: Poor placental function is a common cause of intrauterine growth restriction, which in turn is associated with increased risks of adverse health outcomes. Our prior work suggests that birthweight and childhood obesity-associated genetic variants functionally impact placental function and that placental microRNA are associated with birthweight. To address the influence of the placenta beyond birth, we assessed the relationship between placental microRNAs and early childhood growth. METHODS: Using the SITAR package, we generated two parameters that describe individual weight trajectories of children (0-5 years) in the New Hampshire Birth Cohort Study (NHBCS, n = 238). Using negative binomial generalized linear models, we identified placental microRNAs that relate to growth parameters (FDR < 0.1), while accounting for sex, gestational age at birth, and maternal parity. RESULTS: Genes targeted by the six growth trajectory-associated microRNAs are enriched (FDR < 0.05) in growth factor signaling (TGF/beta: miR-876; EGF/R: miR-155, Let-7c; FGF/R: miR-155; IGF/R: Let-7c, miR-155), calmodulin signaling (miR-216a), and NOTCH signaling (miR-629). CONCLUSIONS: Growth-trajectory microRNAs target pathways affecting placental proliferation, differentiation and function. Our results suggest a role for microRNAs in regulating placental cellular dynamics and supports the Developmental Origins of Health and Disease hypothesis that fetal environment can have impacts beyond birth. IMPACT: We found that growth trajectory associated placenta microRNAs target genes involved in signaling pathways central to the formation, maintenance and function of placenta; suggesting that placental cellular dynamics remain critical to infant growth to term and are under the control of microRNAs. Our results contribute to the existing body of research suggesting that the placenta plays a key role in programming health in the offspring. This is the first study to relate molecular patterns in placenta, specifically microRNAs, to early childhood growth trajectory.

Human placental microRNAs dysregulated by cadmium exposure predict neurobehavioral outcomes at birth.

BACKGROUND: Prenatal cadmium (Cd) exposure has been implicated in both placental toxicity and adverse neurobehavioral outcomes. Placental microRNAs (miRNAs) may function to developmentally program adverse pregnancy and newborn health outcomes in response to gestational Cd exposure. METHODS: In a subset of the Rhode Island Child Health Study (RICHS, n = 115) and the New Hampshire Birth Cohort Study (NHBCS, = 281), we used small RNA sequencing and trace metal analysis to identify Cd-associated expression of placental miRNAs using negative binomial generalized linear models. We predicted mRNAs targeted by Cd-associated miRNAs and relate them to neurobehavioral outcomes at birth through the integration of transcriptomic data and summary scores from the NICU Network Neurobehavioral Scale (NNNS). RESULTS: Placental Cd concentrations are significantly associated with the expression level of five placental miRNAs in NHBCS, with similar effect sizes in RICHS. These miRNA target genes overrepresented in nervous system development, and their expression is correlated with NNNS metrics suggestive of atypical neurobehavioral outcomes at birth. CONCLUSIONS: Gestational Cd exposure is associated with the expression of placental miRNAs. Predicted targets of these miRNAs are involved in nervous system development and may also regulate placental physiology, allowing their dysregulation to modify developmental programming of early life health outcomes. IMPACT: This research aims to address the poor understanding of the molecular mechanisms governing adverse pregnancy and newborn health outcomes in response to Gestational cadmium (Cd) exposure. Our results outline a robust relationship between Cd-associated placental microRNA expression and NICU Network Neurobehavioral Scales (NNNS) at birth indicative of atypical neurobehavior. This study utilized healthy mother-infant cohorts to describe the role of Cd-associated dysregulation of placental microRNAs as a potential mechanism by which adverse neurobehavioral outcomes are developmentally programmed.

Associations between prenatal organophosphate pesticide exposure and placental gene networks.

BACKGROUND: Exposure to organophosphate (OP) pesticides during pregnancy has been linked to deficiencies of neurobehavioral development in childhood; however, the molecular mechanisms underlying this association remain elusive. The placenta plays a crucial role in protecting the fetus from environmental insults and safeguarding proper fetal development including neurodevelopment. The aim of our study is to evaluate changes in the placental transcriptome associated with prenatal OP exposure. METHODS: Pregnant farm workers from two agricultural districts in northern Thailand were recruited for the Study of Asian Women and Offspring's Development and Environmental Exposures (SAWASDEE) from 2017 to 2019. For 254 participants, we measured maternal urinary concentrations of six nonspecific dialkyl phosphates (DAP) metabolites in early, middle, and late pregnancy. In parallel, we profiled the term placental transcriptome from the same participants using RNA-Sequencing and performed Weighted Gene co-expression Network Analysis (WGCNA). Generalized linear regression modeling was used to examine associations of urinary OP metabolites and placental co-expression module eigenvalues. RESULTS: We identified 21 gene co-expression modules in the placenta. From the six DAP metabolites assayed, diethylphosphate (DEP) and diethylthiophosphate (DETP) were detected in more than 70% of the urine samples. Significant associations between DEP at multiple time points and two specific placental gene modules were observed. The 'black' module, enriched in genes involved in epithelial-to-mesenchymal transition (EMT) and hypoxia, was negatively associated with DEP in early (p = 0.034), and late pregnancies (p = 0.016). The 'lightgreen' module, enriched in genes involved in myogenesis and EMT, was negatively associated with DEP in late pregnancy (p = 0.010). We observed 2 hub genes (CELSR1 and PYCR1) of the 'black' module to be negatively associated with DEP in early and late pregnancies. CONCLUSIONS: Our results suggest that prenatal OP exposure may disrupt placental gene networks in a time-dependent manner. Such transcriptomic effects may lead to down-stream changes in placental function that ultimately affect the developing fetus.

Next-generation sequencing of duplication CNVs reveals that most are tandem and some create fusion genes at breakpoints.

Interpreting the genomic and phenotypic consequences of copy-number variation (CNV) is essential to understanding the etiology of genetic disorders. Whereas deletion CNVs lead obviously to haploinsufficiency, duplications might cause disease through triplosensitivity, gene disruption, or gene fusion at breakpoints. The mutational spectrum of duplications has been studied at certain loci, and in some cases these copy-number gains are complex chromosome rearrangements involving triplications and/or inversions. However, the organization of clinically relevant duplications throughout the genome has yet to be investigated on a large scale. Here we fine-mapped 184 germline duplications (14.7 kb-25.3 Mb; median 532 kb) ascertained from individuals referred for diagnostic cytogenetics testing. We performed next-generation sequencing (NGS) and whole-genome sequencing (WGS) to sequence 130 breakpoints from 112 subjects with 119 CNVs and found that most (83%) were tandem duplications in direct orientation. The remainder were triplications embedded within duplications (8.4%), adjacent duplications (4.2%), insertional translocations (2.5%), or other complex rearrangements (1.7%). Moreover, we predicted six in-frame fusion genes at sequenced duplication breakpoints; four gene fusions were formed by tandem duplications, one by two interconnected duplications, and one by duplication inserted at another locus. These unique fusion genes could be related to clinical phenotypes and warrant further study. Although most duplications are positioned head-to-tail adjacent to the original locus, those that are inverted, triplicated, or inserted can disrupt or fuse genes in a manner that might not be predicted by conventional copy-number assays. Therefore, interpreting the genetic consequences of duplication CNVs requires breakpoint-level analysis.

Unbalanced translocations arise from diverse mutational mechanisms including chromothripsis.

Unbalanced translocations are a relatively common type of copy number variation and a major contributor to neurodevelopmental disorders. We analyzed the breakpoints of 57 unique unbalanced translocations to investigate the mechanisms of how they form. Fifty-one are simple unbalanced translocations between two different chromosome ends, and six rearrangements have more than three breakpoints involving two to five chromosomes. Sequencing 37 breakpoint junctions revealed that simple translocations have between 0 and 4 base pairs (bp) of microhomology (n = 26), short inserted sequences (n = 8), or paralogous repeats (n = 3) at the junctions, indicating that translocations do not arise primarily from nonallelic homologous recombination but instead form most often via nonhomologous end joining or microhomology-mediated break-induced replication. Three simple translocations fuse genes that are predicted to produce in-frame transcripts of SIRPG-WWOX, SMOC2-PROX1, and PIEZO2-MTA1, which may lead to gain of function. Three complex translocations have inversions, insertions, and multiple breakpoint junctions between only two chromosomes. Whole-genome sequencing and fluorescence in situ hybridization analysis of two de novo translocations revealed at least 18 and 33 breakpoints involving five different chromosomes. Breakpoint sequencing of one maternally inherited translocation involving four chromosomes uncovered multiple breakpoints with inversions and insertions. All of these breakpoint junctions had 0-4 bp of microhomology consistent with chromothripsis, and both de novo events occurred on paternal alleles. Together with other studies, these data suggest that germline chromothripsis arises in the paternal genome and may be transmitted maternally. Breakpoint sequencing of our large collection of chromosome rearrangements provides a comprehensive analysis of the molecular mechanisms behind translocation formation.

Large inverted duplications in the human genome form via a fold-back mechanism.

Inverted duplications are a common type of copy number variation (CNV) in germline and somatic genomes. Large duplications that include many genes can lead to both neurodevelopmental phenotypes in children and gene amplifications in tumors. There are several models for inverted duplication formation, most of which include a dicentric chromosome intermediate followed by breakage-fusion-bridge (BFB) cycles, but the mechanisms that give rise to the inverted dicentric chromosome in most inverted duplications remain unknown. Here we have combined high-resolution array CGH, custom sequence capture, next-generation sequencing, and long-range PCR to analyze the breakpoints of 50 nonrecurrent inverted duplications in patients with intellectual disability, autism, and congenital anomalies. For half of the rearrangements in our study, we sequenced at least one breakpoint junction. Sequence analysis of breakpoint junctions reveals a normal-copy disomic spacer between inverted and non-inverted copies of the duplication. Further, short inverted sequences are present at the boundary of the disomic spacer and the inverted duplication. These data support a mechanism of inverted duplication formation whereby a chromosome with a double-strand break intrastrand pairs with itself to form a "fold-back" intermediate that, after DNA replication, produces a dicentric inverted chromosome with a disomic spacer corresponding to the site of the fold-back loop. This process can lead to inverted duplications adjacent to terminal deletions, inverted duplications juxtaposed to translocations, and inverted duplication ring chromosomes.

Mouse model implicates GNB3 duplication in a childhood obesity syndrome.

Obesity is a highly heritable condition and a risk factor for other diseases, including type 2 diabetes, cardiovascular disease, hypertension, and cancer. Recently, genomic copy number variation (CNV) has been implicated in cases of early onset obesity that may be comorbid with intellectual disability. Here, we describe a recurrent CNV that causes a syndrome associated with intellectual disability, seizures, macrocephaly, and obesity. This unbalanced chromosome translocation leads to duplication of over 100 genes on chromosome 12, including the obesity candidate gene G protein ß3 (GNB3). We generated a transgenic mouse model that carries an extra copy of GNB3, weighs significantly more than its wild-type littermates, and has excess intraabdominal fat accumulation. GNB3 is highly expressed in the brain, consistent with G-protein signaling involved in satiety and/or metabolism. These functional data connect GNB3 duplication and overexpression to elevated body mass index and provide evidence for a genetic syndrome caused by a recurrent CNV.

Integrating 5-hydroxymethylcytosine into the epigenomic landscape of human embryonic stem cells.

Covalent modification of DNA distinguishes cellular identities and is crucial for regulating the pluripotency and differentiation of embryonic stem (ES) cells. The recent demonstration that 5-methylcytosine (5-mC) may be further modified to 5-hydroxymethylcytosine (5-hmC) in ES cells has revealed a novel regulatory paradigm to modulate the epigenetic landscape of pluripotency. To understand the role of 5-hmC in the epigenomic landscape of pluripotent cells, here we profile the genome-wide 5-hmC distribution and correlate it with the genomic profiles of 11 diverse histone modifications and six transcription factors in human ES cells. By integrating genomic 5-hmC signals with maps of histone enrichment, we link particular pluripotency-associated chromatin contexts with 5-hmC. Intriguingly, through additional correlations with defined chromatin signatures at promoter and enhancer subtypes, we show distinct enrichment of 5-hmC at enhancers marked with H3K4me1 and H3K27ac. These results suggest potential role(s) for 5-hmC in the regulation of specific promoters and enhancers. In addition, our results provide a detailed epigenomic map of 5-hmC from which to pursue future functional studies on the diverse regulatory roles associated with 5-hmC.

Epigenetic landscape of 5-hydroxymethylcytosine and associations with gene expression in placenta.

5-hydroxymethylcystosine (5hmC), is an intermediate product in the DNA demethylation pathway, but may act as a functional epigenetic modification. We have conducted the largest study of site-specific 5hmC in placenta to date using parallel bisulphite and oxidative bisulphite modification with array-based assessment. Incorporating parallel RNA-sequencing data allowed us to assess associations between 5hmC and gene expression, using expression quantitative trait hydroxymethylation (eQTHM) analysis. We identified ~ 47,000 loci with consistently elevated (systematic) 5hmC proportions. Systematic 5hmC was significantly depleted (p < 0.0001) at CpG islands (CGI), and enriched (p < 0.0001) in 'open sea' regions (CpG >4 kb from CGI). 5hmC was most and least abundant at CpGs in enhancers and active transcription start sites (TSS), respectively (p < 0.05). We identified 499 significant (empirical-p <0.05) eQTHMs within 1 MB of the assayed gene. At most (75.4%) eQTHMs, the proportion of 5hmC was positively correlated with transcript abundance. eQTHMs were significantly enriched among enhancer CpGs and depleted among CpGs in active TSS (p < 0.05 for both). Finally, we identified 107 differentially hydroxymethylated regions (DHMRs, p < 0.05) across 100 genes. Our study provides insight into placental distribution of 5hmC, and sheds light on the functional capacity of this epigenetic modification in placenta.

Placental transcriptome variation associated with season, location, and urinary prenatal pyrethroid metabolites of Thai farm-working women.

Prenatal exposure to pyrethroids is linked to adverse health effects in early life and proper placental function is critical to fetal development. This study explores the impact of prenatal pyrethroid exposure, as well as factors impacting exposure and effect, on the placental transcriptome, to understand pyrethroid exposures' relationship to placental function. The study of Asian Women and their Offspring's Development and Environmental Exposures (SAWASDEE) recruited pregnant farm-working women from two agricultural districts in the Chiang Mai province of Thailand between 2017 and 2019. This cohort was predominantly exposed to cypermethrin (type II), alongside pyrethroids such as cyfluthrin (type II) and permethrin (type I). In 253 participants, maternal urinary pyrethroid metabolites, 3-phenoxybenzoic acid (PBA), cis-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (CDCCA), and trans-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (TDCCA) were measured in early, middle, and late pregnancy and adjusted for urinary creatinine. The placental transcriptome was analyzed using RNA-Seq. Using generalized linear regression, we identified differentially expressed genes (DEGs) associated with the sum of each metabolite across pregnancy, as well as those associated with location of residence and season of birth. Pathway and upstream transcription factor analyses were performed to examine potential mechanisms associated with DEGs. Notably, TDCCA and CDCCA levels peaked in late pregnancy, with significant regional differences, particularly higher levels in the Fang region. Placental gene expression analysis showed no DEGs associated with individual metabolites at FDR<0.05. However, 251 DEGs by location, implicating immune response and oxidative phosphorylation pathways, were identified, while season of birth was associated with 2585 DEGs, over-represented in fibrosis signaling and metabolism pathways. Finally, transcription factor analysis identified 226 and 282 transcription factors associated with location and season, respectively, related to cell proliferation, differentiation, and the immune system. These alterations may have significant implications for fetal development and other pathologic processes, highlighting the importance of monitoring environmental exposures during pregnancy.

Diverse mutational mechanisms cause pathogenic subtelomeric rearrangements.

Chromosome rearrangements are a significant cause of intellectual disability and birth defects. Subtelomeric rearrangements, including deletions, duplications and translocations of chromosome ends, were first discovered over 40 years ago and are now recognized as being responsible for several genetic syndromes. Unlike the deletions and duplications that cause some genomic disorders, subtelomeric rearrangements do not typically have recurrent breakpoints and involve many different chromosome ends. To capture the molecular mechanisms responsible for this heterogeneous class of chromosome abnormality, we coupled high-resolution array CGH with breakpoint junction sequencing of a diverse collection of subtelomeric rearrangements. We analyzed 102 breakpoints corresponding to 78 rearrangements involving 28 chromosome ends. Sequencing 21 breakpoint junctions revealed signatures of non-homologous end-joining, non-allelic homologous recombination between interspersed repeats and DNA replication processes. Thus, subtelomeric rearrangements arise from diverse mutational mechanisms. In addition, we find hotspots of subtelomeric breakage at the end of chromosomes 9q and 22q; these sites may correspond to genomic regions that are particularly susceptible to double-strand breaks. Finally, fine-mapping the smallest subtelomeric rearrangements has narrowed the critical regions for some chromosomal disorders.

Effects of prenatal pesticide exposure on the fetal brain and placenta transcriptomes in a rodent model.

Organophosphate and pyrethroid pesticides are among the most extensively used insecticides worldwide. Prenatal exposures to both classes of pesticides have been linked to a wide range of neurobehavioral deficits in the offspring. The placenta is a neuroendocrine organ and the crucial regulator of the intrauterine environment; early-life toxicant exposures could impact neurobehavior by disrupting placental processes. Female C57BL/6 J mice were exposed via oral gavage to an organophosphate, chlorpyrifos (CPF) at 5 mg/kg, a pyrethroid, deltamethrin (DM), at 3 mg/kg, or vehicle only control (CTL). Exposure began two weeks before breeding and continued every three days until euthanasia at gestational day 17. The transcriptomes of fetal brain (CTL n = 18, CPF n = 6, DM n = 8) and placenta (CTL n = 19, CPF n = 16, DM n = 12) were obtained through RNA sequencing, and resulting data was evaluated using weighted gene co-expression networks, differential expression, and pathway analyses. Fourteen brain gene co-expression modules were identified; CPF exposure disrupted the module related to ribosome and oxidative phosphorylation, whereas DM disrupted the modules related to extracellular matrix and calcium signaling. In the placenta, network analyses revealed 12 gene co-expression modules. While CPF exposure disrupted modules related to endocytosis, Notch and Mapk signaling, DM exposure dysregulated modules linked to spliceosome, lysosome and Mapk signaling pathways. Overall, in both tissues, CPF exposure impacted oxidative phosphorylation, while DM was linked to genes involved in spliceosome and cell cycle. The transcription factor Max involved in cell proliferation was overexpressed by both pesticides in both tissues. In summary, gestational exposure to two different classes of pesticide can induce similar pathway-level transcriptome changes in the placenta and the brain; further studies should investigate if these changes are linked to neurobehavioral impairments.

Variation in placental microRNA expression associates with maternal family history of cardiovascular disease.

In the United States, cardiovascular disease is the leading cause of death and the rate of maternal mortality remains among the highest of any industrialized nation. Maternal cardiometabolic health throughout gestation and postpartum is representative of placental health and physiology. Both proper placental functionality and placental microRNA expression are essential to successful pregnancy outcomes, and both are highly sensitive to genetic and environmental sources of variation. Placental pathologies, such as preeclampsia, are associated with maternal cardiovascular health but may also contribute to the developmental programming of chronic disease in offspring. However, the role of more subtle alterations to placental function and microRNA expression in this developmental programming remains poorly understood. We performed small RNA sequencing to investigate microRNA in placentae from the Rhode Island Child Health Study (n = 230). MicroRNA counts were modeled on maternal family history of cardiovascular disease using negative binomial generalized linear models. MicroRNAs were considered to be differentially expressed at a false discovery rate (FDR) less than 0.10. Parallel mRNA sequencing data and bioinformatic target prediction software were then used to identify potential mRNA targets of differentially expressed microRNAs. Nine differentially expressed microRNAs were identified (FDR < 0.1). Bioinformatic target prediction revealed 66 potential mRNA targets of these microRNAs, many of which are implicated in TGFß signaling pathway but also in pathways involving cellular metabolism and immunomodulation. A robust association exists between familial cardiovascular disease and placental microRNA expression which may be implicated in both placental insufficiencies and the developmental programming of chronic disease.

Prenatal lead (Pb) exposure is associated with differential placental DNA methylation and hydroxymethylation in a human population.

Prenatal lead (Pb) exposure is associated with adverse developmental outcomes and to epigenetic alterations such as DNA methylation and hydroxymethylation in animal models and in newborn blood. Given the importance of the placenta in foetal development, we sought to examine how prenatal Pb exposure was associated with differential placental DNA methylation and hydroxymethylation and to identify affected biological pathways linked to developmental outcomes. Maternal (n = 167) and infant (n = 172) toenail and placenta (n = 115) samples for prenatal Pb exposure were obtained from participants in a US birth cohort, and methylation and hydroxymethylation data were quantified using the Illumina Infinium MethylationEPIC BeadChip. An epigenome-wide association study was applied to identify differential methylation and hydroxymethylation associated with Pb exposure. Biological functions of the Pb-associated genes were determined by overrepresentation analysis through ConsensusPathDB. Prenatal Pb quantified from maternal toenail, infant toenail, and placenta was associated with 480, 27, and 2 differentially methylated sites (q < 0.05), respectively, with both increases and decreases associated with exposure. Alternatively, we identified 2, 1, and 14 differentially hydroxymethylated site(s) associated with maternal toenail, infant toenail, and placental Pb, respectively, with most showing increases in hydroxymethylation with exposure. Significantly overrepresented pathways amongst genes associated with differential methylation and hydroxymethylation (q < 0.10) included mechanisms pertaining to nervous system and organ development, calcium transport and regulation, and signalling activities. Our results suggest that both methylation and hydroxymethylation in the placenta can be variable based on Pb exposure and that the pathways impacted could affect placental function.

Selenium-associated differentially expressed microRNAs and their targeted mRNAs across the placental genome in two U.S. birth cohorts.

Selenium is an important micronutrient for foetal development. MicroRNAs play an important role in the function of the placenta, in communication between the placenta and maternal systems, and their expression can be altered through environmental and nutritional cues. To investigate the associations between placental selenium concentration and microRNA expression in the placenta, our observational study included 393 mother-child pairs from the New Hampshire Birth Cohort Study (NHBCS) and the Rhode Island Child Health Study (RICHS). Placental selenium concentrations were quantified using inductively coupled plasma mass spectrometry, and microRNA transcripts were measured using RNA-seq. We fit negative binomial additive models for assessing the association between selenium and microRNAs. We used the microRNA Data Integration Portal (mirDIP) to predict the target mRNAs of the differentially expressed microRNAs and verified the relationships between miRNA and mRNA targets in a subset of samples using existing whole transcriptome data (N = 199). We identified a non-monotonic association between selenium concentration and the expression of miR-216a-5p/miR-217-5p cluster (effective degrees of freedom, EDF = 2.44 and 2.08; FDR = 3.08 × 10-5) in placenta. Thirty putative target mRNAs of miR-216a-5p and/or miR-217-5p were identified computationally and empirically and were enriched in selenium metabolic pathways (driven by selenoprotein coding genes, TXNRD2 and SELENON). Our findings suggest that selenium influences placental microRNA expression. Further, miR-216a-5p and its putative target mRNAs could be the potential mechanistic targets of the health effect of selenium.

Developmental chronodisruption alters placental signaling in mice.

Chronodisruption has been largely overlooked as a developmental exposure. The placenta, a conduit between the maternal and fetal environments, may relay circadian cues to the fetus. We have previously shown that developmental chronodisruption causes visual impairment and increased retinal microglial and macrophage marker expression. Here, we investigated the impacts of environmental chronodisruption on fetal and placental outcomes in a C57BL/6J mouse (Mus musculus) model. Developmental chronodisruption had no effect on embryo count, placental weight, or fetal sex ratio. When measured with RNAseq, mice exposed to developmental chronodisruption (CD) had differential placental expression of several transcripts including Serpinf1, which encodes pigment epithelium-derived factor (PEDF). Immunofluorescence of microglia/macrophage markers, Iba1 and CD11b, also revealed significant upregulation of immune cell markers in CD-exposed placenta. Our results suggest that in utero chronodisruption enhances placental immune cell expression, potentially programming a pro-inflammatory tissue environment.

Placental microRNA expression associates with birthweight through control of adipokines: results from two independent cohorts.

MicroRNAs are non-coding RNAs that regulate gene expression post-transcriptionally. In the placenta, the master regulator of foetal growth and development, microRNAs shape the basic processes of trophoblast biology and specific microRNA have been associated with foetal growth. To comprehensively assess the role of microRNAs in placental function and foetal development, we have performed small RNA sequencing to profile placental microRNAs from two independent mother-infant cohorts: the Rhode Island Child Health Study (n = 225) and the New Hampshire Birth Cohort Study (n = 317). We modelled microRNA counts on infant birthweight percentile (BWP) in each cohort, while accounting for race, sex, parity, and technical factors, using negative binomial generalized linear models. We identified microRNAs that were differentially expressed (DEmiRs) with BWP at false discovery rate (FDR) less than 0.05 in both cohorts. hsa-miR-532-5p (miR-532) was positively associated with BWP in both cohorts. By integrating parallel whole transcriptome and small RNA sequencing in the RICHS cohort, we identified putative targets of miR-532. These targets are enriched for pathways involved in adipogenesis, adipocytokine signalling, energy metabolism, and hypoxia response, and included Leptin, which we further demonstrated to have a decreasing expression with increasing BWP, particularly in male infants. Overall, we have shown a robust and reproducible association of miR-532 with BWP, which could influence BWP through regulation of adipocytokines Leptin and Adiponectin.

Longitudinal changes in epigenetic age in youth with perinatally acquired HIV and youth who are perinatally HIV-exposed uninfected.

OBJECTIVES: To quantify the rate of change in epigenetic age compared with chronological age over time in youth with perinatally acquired HIV (YPHIV) and youth who are perinatally HIV-exposed uninfected (YPHEU). DESIGN: Longitudinal study of 32 YPHIV and 8 YPHEU with blood samples collected at two time points at least 3 years apart. METHODS: DNA methylation was measured using the Illumina MethylationEPIC array and epigenetic age was calculated using the Horvath method. Linear mixed effects models were fit to estimate the average change in epigenetic age for a 1-year change in chronological age separately for YPHIV and YPHEU. RESULTS: Median age was 10.9 and 16.8 years at time 1 and 2, respectively. Groups were balanced by sex (51% male) and race (67% black). Epigenetic age increased by 1.23 years (95% CI 1.03--1.43) for YPHIV and 0.95 years (95% CI 0.74--1.17) for YPHEU per year increase in chronological age. Among YPHIV, in a model with chronological age, a higher area under the curve (AUC) viral load was associated with an increase in epigenetic age over time [2.19 years per log10 copies/ml, (95% CI 0.65--3.74)], whereas a higher time-averaged AUC CD4+ T-cell count was associated with a decrease in epigenetic age over time [-0.34 years per 100 cells/µl, (95% CI -0.63 to -0.06)] in YPHIV. CONCLUSION: We observed an increase in the rate of epigenetic aging over time in YPHIV, but not in YPHEU. In YPHIV, higher viral load and lower CD4+ T-cell count were associated with accelerated epigenetic aging, emphasizing the importance of early and sustained suppressive treatment for YPHIV, who will receive lifelong ART.