Identifying Reproducible Transcription Regulator Coexpression Patterns with Single Cell Transcriptomics.

The proliferation of single cell transcriptomics has potentiated our ability to unveil patterns that reflect dynamic cellular processes, rather than cell type compositional effects that emerge from bulk tissue samples. In this study, we leverage a broad collection of single cell RNA-seq data to identify the gene partners whose expression is most coordinated with each human and mouse transcription regulator (TR). We assembled 120 human and 103 mouse scRNA-seq datasets from the literature (>28 millions cells), constructing a single cell coexpression network for each. We aimed to understand the consistency of TR coexpression profiles across a broad sampling of biological contexts, rather than examine the preservation of context-specific signals. Our workflow therefore explicitly prioritizes the patterns that are most reproducible across cell types. Towards this goal, we characterize the similarity of each TR's coexpression within and across species. We create single cell coexpression rankings for each TR, demonstrating that this aggregated information recovers literature curated targets on par with ChIP-seq data. We then combine the coexpression and ChIP-seq information to identify candidate regulatory interactions supported across methods and species. Finally, we highlight interactions for the important neural TR ASCL1 to demonstrate how our compiled information can be adopted for community use.

Characterizing the targets of transcription regulators by aggregating ChIP-seq and perturbation expression data sets.

Mapping the gene targets of chromatin-associated transcription regulators (TRs) is a major goal of genomics research. ChIP-seq of TRs and experiments that perturb a TR and measure the differential abundance of gene transcripts are a primary means by which direct relationships are tested on a genomic scale. It has been reported that there is a poor overlap in the evidence across gene regulation strategies, emphasizing the need for integrating results from multiple experiments. Although research consortia interested in gene regulation have produced a valuable trove of high-quality data, there is an even greater volume of TR-specific data throughout the literature. In this study, we show a workflow for the identification, uniform processing, and aggregation of ChIP-seq and TR perturbation experiments for the ultimate purpose of ranking human and mouse TR-target interactions. Focusing on an initial set of eight regulators (ASCL1, HES1, MECP2, MEF2C, NEUROD1, PAX6, RUNX1, and TCF4), we identified 497 experiments suitable for analysis. We used this corpus to examine data concordance, to identify systematic patterns of the two data types, and to identify putative orthologous interactions between human and mouse. We build upon commonly used strategies to forward a procedure for aggregating and combining these two genomic methodologies, assessing these rankings against independent literature-curated evidence. Beyond a framework extensible to other TRs, our work also provides empirically ranked TR-target listings, as well as transparent experiment-level gene summaries for community use.

Human amnionic progenitor cell secretome mitigates the consequence of traumatic optic neuropathy in a mouse model.

Traumatic optic neuropathy (TON) is a condition in which acute injury to the optic nerve from direct or indirect trauma results in vision loss. The most common cause of TON is indirect injury to the optic nerve caused by concussive forces that are transmitted to the optic nerve. TON occurs in up to 5% of closed-head trauma patients and there is currently no known effective treatment. One potential treatment option for TON is ST266, a cell-free biological solution containing the secretome of amnion-derived multipotent progenitor (AMP) cells. We investigated the efficacy of intranasal ST266 in a mouse model of TON induced by blunt head trauma. Injured mice treated with a 10-day regimen of ST266 showed an improvement in spatial memory and learning, a significant preservation of retinal ganglion cells, and a decrease in neuropathological markers in the optic nerve, optic tract, and dorsal lateral geniculate nucleus. ST266 treatment effectively downregulated the NLRP3 inflammasome-mediated neuroinflammation pathway after blunt trauma. Overall, treatment with ST266 was shown to improve functional and pathological outcomes in a mouse model of TON, warranting future exploration of ST266 as a cell-free therapeutic candidate for testing in all optic neuropathies.

Impact of gulf war toxic exposures after mild traumatic brain injury.

Chemical and pharmaceutical exposures have been associated with the development of Gulf War Illness (GWI), but how these factors interact with the pathophysiology of traumatic brain injury (TBI) remains an area of study that has received little attention thus far. We studied the effects of pyridostigmine bromide (an anti-nerve agent) and permethrin (a pesticide) exposure in a mouse model of repetitive mild TBI (r-mTBI), with 5 impacts over a 9-day period, followed by Gulf War (GW) toxicant exposure for 10 days beginning 30 days after the last head injury. We then assessed the chronic behavioral and pathological sequelae 5 months after GW agent exposure. We observed that r-mTBI and GWI cumulatively affect the spatial memory of mice in the Barnes maze and result in a shift of search strategies employed by r-mTBI/GW exposed mice. GW exposure also produced anxiety-like behavior in sham animals, but r-mTBI produced disinhibition in both the vehicle and GW treated mice. Pathologically, GW exposure worsened r-mTBI dependent axonal degeneration and neuroinflammation, increased oligodendrocyte cell counts, and increased r-mTBI dependent phosphorylated tau, which was found to colocalize with oligodendrocytes in the corpus callosum. These results suggest that GW exposures may worsen TBI-related deficits. Veterans with a history of both GW chemical exposures as well as TBI may be at higher risk for worse symptoms and outcomes. Subsequent exposure to various toxic substances can influence the chronic nature of mTBI and should be considered as an etiological factor influencing mTBI recovery.

Subacute and chronic proteomic and phosphoproteomic analyses of a mouse model of traumatic brain injury at two timepoints and comparison with chronic traumatic encephalopathy in human samples.

Repetitive mild traumatic brain injury (r-mTBI) is the most widespread type of brain trauma worldwide. The cumulative injury effect triggers long-lasting pathological and molecular changes that may increase risk of chronic neurodegenerative diseases. R-mTBI is also characterized by changes in the brain proteome, where the majority of molecules altered early post-TBI are different from those altered at more chronic phases. This differentiation may contribute to the heterogeneity of available data on potential therapeutic targets and may present an obstacle in developing effective treatments. Here, we aimed to characterize a proteome profile of r-mTBI in a mouse model at two time points - 3 and 24 weeks post last TBI, as this may be a more relevant therapeutic window for individuals suffering negative consequences of r-mTBI. We identified a great number of proteins and phosphoproteins that remain continuously dysregulated from 3 to 24 weeks. These proteins may serve as effective therapeutic targets for sub-acute and chronic stages of post r-mTBI. We also compared canonical pathway activation associated with either total proteins or phosphoproteins and revealed that they both are upregulated at 24 weeks. However, at 3 weeks post-TBI, only pathways associated with total proteins are upregulated, while pathways driven by phosphoproteins are downregulated. Finally, to assess the translatability of our data, we compared proteomic changes in our mouse model with those reported in autopsied human samples of Chronic Traumatic Encephalopathy (CTE) patients compared to controls. We observed 39 common proteins that were upregulated in both species and 24 common pathways associated with these proteins. These findings support the translational relevance of our mouse model of r-mTBI for successful identification and translation of therapeutic targets.

Experiment level curation of transcriptional regulatory interactions in neurodevelopment.

To facilitate the development of large-scale transcriptional regulatory networks (TRNs) that may enable in-silico analyses of disease mechanisms, a reliable catalogue of experimentally verified direct transcriptional regulatory interactions (DTRIs) is needed for training and validation. There has been a long history of using low-throughput experiments to validate single DTRIs. Therefore, we reason that a reliable set of DTRIs could be produced by curating the published literature for such evidence. In our survey of previous curation efforts, we identified the lack of details about the quantity and the types of experimental evidence to be a major gap, despite the theoretical importance of such details for the identification of bona fide DTRIs. We developed a curation protocol to inspect the published literature for support of DTRIs at the experiment level, focusing on genes important to the development of the mammalian nervous system. We sought to record three types of low-throughput experiments: Transcription factor (TF) perturbation, TF-DNA binding, and TF-reporter assays. Using this protocol, we examined a total of 1,310 papers to assemble a collection of 1,499 unique DTRIs, involving 251 TFs and 825 target genes, many of which were not reported in any other DTRI resource. The majority of DTRIs (965; 64%) were supported by two or more types of experimental evidence and 27% were supported by all three. Of the DTRIs with all three types of evidence, 170 had been tested using primary tissues or cells and 44 had been tested directly in the central nervous system. We used our resource to document research biases among reports towards a small number of well-studied TFs. To demonstrate a use case for this resource, we compared our curation to a previously published high-throughput perturbation screen and found significant enrichment of the curated targets among genes differentially expressed in the developing brain in response to Pax6 deletion. This study demonstrates a proof-of-concept for the assembly of a high resolution DTRI resource to support the development of large-scale TRNs.

Maternal psychosocial risk factors and child gestational epigenetic age in a South African birth cohort study.

Accelerated epigenetic aging relative to chronological age has been found to be associated with higher risk of mortality in adults. However, little is known about whether and how in utero exposures might shape child gestational epigenetic age (EA) at birth. We aimed to explore associations between maternal psychosocial risk factors and deviation in child gestational EA at birth (i.e., greater or lower EA relative to chronological age) in a South African birth cohort study-the Drakenstein Child Health Study. Maternal psychosocial risk factors included trauma/stressor exposure; posttraumatic stress disorder (PTSD); depression; psychological distress; and alcohol/tobacco use. Child gestational EA at birth was calculated using an epigenetic clock previously devised for neonates; and gestational EA deviation was calculated as the residuals of the linear model between EA and chronological gestational age. Bivariate linear regression was then used to explore unadjusted associations between maternal/child risk factors and child gestational EA residuals at birth. Thereafter, a multivariable regression method was used to determine adjusted associations. Data from 271 maternal-child dyads were included in the current analysis. In the multivariable regression model, maternal PTSD was significantly and negatively associated with child gestational EA residuals at birth (ß = -1.95; p = 0.018), controlling for study site, sex of the child, head circumference at birth, birthweight, mode of delivery, maternal estimated household income, body mass index (BMI) at enrolment, HIV status, anaemia, psychological distress, and prenatal tobacco or alcohol use. Given the novelty of this preliminary finding, and its potential translational relevance, further studies to delineate underlying biological pathways and to explore clinical implications of EA deviation are warranted.

A 3-month-delayed treatment with anatabine improves chronic outcomes in two different models of repetitive mild traumatic brain injury in hTau mice.

To date, an overwhelming number of preclinical studies have addressed acute treatment in mild TBI (mTBI) and repetitive mTBI (r-mTBI), whereas, in humans, there often exists a significant time gap between the injury and the first medical intervention. Our study focused on a delayed treatment with anatabine, an anti-inflammatory compound, in hTau mice using two different models of r-mTBI. The rationale for using two models of the same impact but different frequencies (5 hit mTBI over 9 days and 24 hit mTBI over 90 days) was chosen to address the heterogeneity of r-mTBI in clinical population. Following the last injury in each model, three months elapsed before the initiation of treatment. Anatabine was administered in drinking water for 3 months thereafter. Our data demonstrated that a 3-month delayed treatment with anatabine mitigated astrogliosis in both TBI paradigms but improved cognitive functions only in more-frequently-injured mice (24 hit mTBI). We also found that anatabine decreased the phosphorylation of tau protein and NFκB, which were increased after r-mTBI in both models. The ability of anatabine to suppress these mechanisms suggests that delayed treatment can be effective for clinical population of r-mTBI. The discrepancy between the two models with regard to changes in cognitive performance suggests that r-mTBI heterogeneity may influence treatment efficiency and should be considered in therapeutic development.

Methylation of cysteinyl leukotriene receptor 1 genes associates with lung function in asthmatics exposed to traffic-related air pollution.

Air pollution is associated with early declines in lung function and increased levels of asthma-related cysteinyl leukotrienes (CysLT) but a biological pathway linking this rapid response has not been delineated. In this randomized controlled diesel exhaust (DE) challenge study of 16 adult asthmatics, increased exposure-attributable urinary leukotriene E4 (uLTE4, a biomarker of cysteinyl leukotriene production) was correlated (p = 0.04) with declines in forced expiratory volume in 1-second (FEV1) within 6 hours of exposure. Exposure-attributable uLTE4 increases were correlated (p = 0.02) with increased CysLT receptor 1 (CysLTR1) methylation in peripheral blood mononuclear cells which, in turn, was marginally correlated (p = 0.06) with decreased CysLTR1 expression. Decreased CysLTR1 expression was, in turn, correlated (p = 0.0007) with FEV1 declines. During the same time period, increased methylation of GPR17 (a negative regulator of CysLTR1) was observed after DE exposure (p = 0.02); this methylation increase was correlated (p = 0.001) with decreased CysLTR1 methylation which, in turn, was marginally correlated (p = 0.06) with increased CysLTR1 expression; increased CysLTR1 expression was correlated (p = 0.0007) with FEV1 increases. Collectively, these data delineate a potential mechanistic pathway linking increased DE exposure-attributable CysLT levels to lung function declines through changes in CysLTR1-related methylation and gene expression.

Nilvadipine suppresses inflammation via inhibition of P-SYK and restores spatial memory deficits in a mouse model of repetitive mild TBI.

Repeated exposure to mild TBI (mTBI) has been linked to an increased risk of Alzheimer's disease (AD), chronic traumatic encephalopathy (CTE) and other neurodegenerative diseases. Some pathological features typically observed in AD have been found in postmortem brains of TBI and CTE, hence treatments tested for AD have a potential to be effective against r-mTBI outcomes. Neuroinflammation may present a possible answer due to its central role both in acute brain injury and in chronic degenerative-like disorders. Our previous studies have shown that drug nilvadipine, acting as an inhibitor of spleen tyrosine kinase (SYK), is effective at reducing inflammation, tau hyperphosphorylation and amyloid production in AD mouse models. To demonstrate the effect of nilvadipine in the absence of age-related variables, we introduced the same treatment to young r-mTBI mice. We further investigate therapeutic mechanisms of nilvadipine using its racemic properties. Both enantiomers, (+)-nilvadipine and (-)-nilvadipine, can lower SYK activity, whereas (+)-nilvadipine is also a potent L-type calcium channel blocker (CCB) and shown to be anti-hypertensive. All r-mTBI mice exhibited increased neuroinflammation and impaired cognitive performance and motor functions. Treatment with racemic nilvadipine mitigated the TBI-induced inflammatory response and significantly improved spatial memory, whereas (-)-enantiomer decreased microgliosis and improved spatial memory but failed to reduce the astroglial response to as much as the racemate. These results suggest the therapeutic potential of SYK inhibition that is enhanced when combined with the CCB effect, which indicate a therapeutic advantage of multi-action drugs for r-mTBI.

Parenting Interacts With Plasticity Genes in Predicting Behavioral Outcomes in Preschoolers.

BACKGROUND: Public health and pediatric nurses typically focus on supporting parenting to reduce the likelihood of children's behavioral problems. Studies have identified interactions between early exposures to stress in caregiving and child genotype in predicting children's behavioral problems, such that certain genotypes connote greater differential susceptibility or plasticity to environmental stressors. We sought to uncover the interaction between observational measures of parent-child relationship quality and genotype in predicting early-onset behavioral problems in 24-month-olds, using prospective methods. METHODS: We conducted a secondary analysis of data collected on a subsample of 176 women and their infants enrolled during pregnancy in the ongoing Alberta Pregnancy Outcomes and Nutrition (APrON) cohort study. Inclusion criteria required mothers to be ≥18 years of age, English speaking and ≤22 weeks gestational age at enrollment. Genetic data were obtained from blood leukocytes and buccal epithelial cell samples, collected from infants at three months of age. For each child, the presence of plasticity alleles was determined for BDNF, CNR1, DRD2/ANKK1, DRD4, DAT1, 5-HTTLPR, and MAOA and an overall index was calculated to summarize the number of plasticity alleles present. Observational assessments of parent-child relationship quality (sensitivity, controlling, and unresponsiveness) were conducted at six months of age. Children's internalizing (e.g., emotionally reactive, anxious/depressed, somatic complaint, withdrawn) and externalizing (e.g., aggression, inattention) behaviors were assessed at 24 months of age. After extracting genetic data, a maximum likelihood method for regressions was employed with Akaike Information Criterion (AIC) for model selection. RESULTS: When parents were less responsive and children possessed more plasticity alleles, children were more likely to be emotionally reactive, anxious/depressed, report somatic complaints, and withdrawn, while when parents were less responsive and children possessed fewer plasticity alleles, children were less likely to display these internalizing behaviors, in a differentially susceptible manner. Furthermore, when parents were more responsive, and children possessed more plasticity alleles, children were less likely to display internalizing behaviors (P = 0.034). Similarly, children who possessed either the CNR1-A plasticity allele (P = 0.010) or DAT1 9-repeat plasticity allele (P = 0.036) and experienced more/less parental control displayed more/fewer externalizing problems, respectively, in a differentially susceptible manner. CONCLUSIONS: The plasticity index score interacted with parental unresponsiveness in predicting anxiety and depressive behavioral problems in children, while individual genetic variants interacted with parental controlling behavior in predicting aggression and inattention in children, suggestive of differential susceptibility to caregiving. Especially in the context of nursing interventions designed to support childrearing and children's development, nurses need to be aware of the interactions between child genotype and parenting in understanding how well interventions will work in promoting optimal child behavior.

The PedBE clock accurately estimates DNA methylation age in pediatric buccal cells.

The development of biological markers of aging has primarily focused on adult samples. Epigenetic clocks are a promising tool for measuring biological age that show impressive accuracy across most tissues and age ranges. In adults, deviations from the DNA methylation (DNAm) age prediction are correlated with several age-related phenotypes, such as mortality and frailty. In children, however, fewer such associations have been made, possibly because DNAm changes are more dynamic in pediatric populations as compared to adults. To address this gap, we aimed to develop a highly accurate, noninvasive, biological measure of age specific to pediatric samples using buccal epithelial cell DNAm. We gathered 1,721 genome-wide DNAm profiles from 11 different cohorts of typically developing individuals aged 0 to 20 y old. Elastic net penalized regression was used to select 94 CpG sites from a training dataset (n = 1,032), with performance assessed in a separate test dataset (n = 689). DNAm at these 94 CpG sites was highly predictive of age in the test cohort (median absolute error = 0.35 y). The Pediatric-Buccal-Epigenetic (PedBE) clock was characterized in additional cohorts, showcasing the accuracy in longitudinal data, the performance in nonbuccal tissues and adult age ranges, and the association with obstetric outcomes. The PedBE tool for measuring biological age in children might help in understanding the environmental and contextual factors that shape the DNA methylome during child development, and how it, in turn, might relate to child health and disease.

Treatment With Nilvadipine Mitigates Inflammatory Pathology and Improves Spatial Memory in Aged hTau Mice After Repetitive Mild TBI.

Mild traumatic brain injury (mTBI) is the most common form of brain trauma worldwide. The effects of mTBI are not well-studied within the elderly population, yet older adults constitute a significant portion of all mTBI patients. Few preclinical studies have focused on the effects of mTBI, or mTBI treatments, in the aged brain, and none have explored repetitive mTBI (r-mTBI). In this study, we have administered our well-characterized 5-injury model (5 r-mTBI) to hTau mice aged 24 months to explore the neurobehavioral and neuropathological outcomes, and the effects of treatment with the dihydropyridine, Nilvadipine. Our previous studies have shown that Nilvadipine inhibits spleen tyrosine kinase (Syk), is effective at reducing inflammation, tau hyperphosphorylation, and amyloid production, and it has recently been investigated in a European Phase III clinical trial for Alzheimer's disease (AD). In our 24-month-old r-mTBI mice, we observed increased neuroinflammation and a trend toward impaired cognitive performance compared to sham controls. Treatment with Nilvadipine mitigated the TBI-induced inflammatory response in aged r-mTBI animals and significantly improved spatial memory. To our knowledge, this is the only preclinical study focusing on the treatment of r-mTBI in aged, and these results suggest a therapeutic potential of Nilvadipine for consequences of mTBI.

Systematic evaluation of DNA methylation age estimation with common preprocessing methods and the Infinium MethylationEPIC BeadChip array.

BACKGROUND: The capacity of technologies measuring DNA methylation (DNAm) is rapidly evolving, as are the options for applicable bioinformatics methods. The most commonly used DNAm microarray, the Illumina Infinium HumanMethylation450 (450K array), has recently been replaced by the Illumina Infinium HumanMethylationEPIC (EPIC array), nearly doubling the number of targeted CpG sites. Given that a subset of 450K CpG sites is absent on the EPIC array and that several tools for both data normalization and analyses were developed on the 450K array, it is important to assess their utility when applied to EPIC array data. One of the most commonly used 450K tools is the pan-tissue epigenetic clock, a multivariate predictor of biological age based on DNAm at 353 CpG sites. Of these CpGs, 19 are missing from the EPIC array, thus raising the question of whether EPIC data can be used to accurately estimate DNAm age. We also investigated a 71-CpG epigenetic age predictor, referred to as the Hannum method, which lacks 6 probes on the EPIC array. To evaluate these epigenetic clocks in EPIC data properly, a prior assessment of the effects of data preprocessing methods on DNAm age is also required. METHODS: DNAm was quantified, on both the 450K and EPIC platforms, from human primary monocytes derived from 172 individuals. We calculated DNAm age from raw, and three different preprocessed data forms to assess the effects of different processing methods on the DNAm age estimate. Using an additional cohort, we also investigated DNAm age of peripheral blood mononuclear cells, bronchoalveolar lavage, and bronchial brushing samples using the EPIC array. RESULTS: Using monocyte-derived data from subjects on both the 450K and EPIC, we found that DNAm age was highly correlated across both raw and preprocessing methods (r > 0.91). Thus, the correlation between chronological age and the DNAm age estimate is largely unaffected by platform differences and normalization methods. However, we found that the choice of normalization method and measurement platform can lead to a systematic offset in the age estimate which in turn leads to an increase in the median error. Comparing the 450K and EPIC DNAm age estimates, we observed that the median absolute difference was 1.44-3.10 years across preprocessing methods. CONCLUSIONS: Here, we have provided evidence that the epigenetic clock is resistant to the lack of 19 CpG sites missing from the EPIC array as well as highlighted the importance of considering the technical variance of the epigenetic when interpreting group differences below the reported error. Furthermore, our study highlights the utility of epigenetic age acceleration measure, the residuals from a linear regression of DNAm age on chronological age, as the resulting values are robust with respect to normalization methods and measurement platforms.

Impact of age on acute post-TBI neuropathology in mice expressing humanized tau: a Chronic Effects of Neurotrauma Consortium Study.

OBJECTIVES: We hypothesized that polypathology is more severe in older than younger mice during the acute phase following repetitive mild traumatic brain injury (r-mTBI). METHODS: Young and aged male and female mice transgenic for human tau (hTau) were exposed to r-mTBI or a sham procedure. Twenty-four hours post-last injury, mouse brain tissue was immunostained for alterations in astrogliosis, microgliosis, tau pathology, and axonal injury. RESULTS: Quantitative analysis revealed a greater percent distribution of glial fibrillary acid protein and Iba-1 reactivity in the brains of all mice exposed to r-mTBI compared to sham controls. With respect to axonal injury, the number of amyloid precursor protein-positive profiles was increased in young vs aged mice post r-mTBI. An increase in tau immunoreactivity was found in young and aged injured male hTau mice. CONCLUSIONS: We report the first evidence in our model that r-mTBI precipitates a complex sequelae of events in aged vs young hTau mice at an acute time point, typified by an increase in phosphorylated tau and astroglisosis, and a diminished microgliosis response and axonal injury in aged mice. These findings suggest differential age-dependent effects in TBI pathobiology.

DNA methylation as a predictor of fetal alcohol spectrum disorder.

Background: Fetal alcohol spectrum disorder (FASD) is a developmental disorder that manifests through a range of cognitive, adaptive, physiological, and neurobiological deficits resulting from prenatal alcohol exposure. Although the North American prevalence is currently estimated at 2-5%, FASD has proven difficult to identify in the absence of the overt physical features characteristic of fetal alcohol syndrome. As interventions may have the greatest impact at an early age, accurate biomarkers are needed to identify children at risk for FASD. Building on our previous work identifying distinct DNA methylation patterns in children and adolescents with FASD, we have attempted to validate these associations in a different clinical cohort and to use our DNA methylation signature to develop a possible epigenetic predictor of FASD. Methods: Genome-wide DNA methylation patterns were analyzed using the Illumina HumanMethylation450 array in the buccal epithelial cells of a cohort of 48 individuals aged 3.5-18 (24 FASD cases, 24 controls). The DNA methylation predictor of FASD was built using a stochastic gradient boosting model on our previously published dataset FASD cases and controls (GSE80261). The predictor was tested on the current dataset and an independent dataset of 48 autism spectrum disorder cases and 48 controls (GSE50759). Results: We validated findings from our previous study that identified a DNA methylation signature of FASD, replicating the altered DNA methylation levels of 161/648 CpGs in this independent cohort, which may represent a robust signature of FASD in the epigenome. We also generated a predictive model of FASD using machine learning in a subset of our previously published cohort of 179 samples (83 FASD cases, 96 controls), which was tested in this novel cohort of 48 samples and resulted in a moderately accurate predictor of FASD status. Upon testing the algorithm in an independent cohort of individuals with autism spectrum disorder, we did not detect any bias towards autism, sex, age, or ethnicity. Conclusion: These findings further support the association of FASD with distinct DNA methylation patterns, while providing a possible entry point towards the development of epigenetic biomarkers of FASD.

Choice of surrogate tissue influences neonatal EWAS findings.

BACKGROUND: Epigenomes are tissue specific and thus the choice of surrogate tissue can play a critical role in interpreting neonatal epigenome-wide association studies (EWAS) and in their extrapolation to target tissue. To develop a better understanding of the link between tissue specificity and neonatal EWAS, and the contributions of genotype and prenatal factors, we compared genome-wide DNA methylation of cord tissue and cord blood, two of the most accessible surrogate tissues at birth. METHODS: In 295 neonates, DNA methylation was profiled using Infinium HumanMethylation450 beadchip arrays. Sites of inter-individual variability in DNA methylation were mapped and compared across the two surrogate tissues at birth, i.e., cord tissue and cord blood. To ascertain the similarity to target tissues, DNA methylation profiles of surrogate tissues were compared to 25 primary tissues/cell types mapped under the Epigenome Roadmap project. Tissue-specific influences of genotype on the variable CpGs were also analyzed. Finally, to interrogate the impact of the in utero environment, EWAS on 45 prenatal factors were performed and compared across the surrogate tissues. RESULTS: Neonatal EWAS results were tissue specific. In comparison to cord blood, cord tissue showed higher inter-individual variability in the epigenome, with a lower proportion of CpGs influenced by genotype. Both neonatal tissues were good surrogates for target tissues of mesodermal origin. They also showed distinct phenotypic associations, with effect sizes of the overlapping CpGs being in the same order of magnitude. CONCLUSIONS: The inter-relationship between genetics, prenatal factors and epigenetics is tissue specific, and requires careful consideration in designing and interpreting future neonatal EWAS. TRIAL REGISTRATION: This birth cohort is a prospective observational study, designed to study the developmental origins of health and disease, and was retrospectively registered on 1 July 2010 under the identifier NCT01174875 .

Maternal blood contamination of collected cord blood can be identified using DNA methylation at three CpGs.

BACKGROUND: Cord blood is a commonly used tissue in environmental, genetic, and epigenetic population studies due to its ready availability and potential to inform on a sensitive period of human development. However, the introduction of maternal blood during labor or cross-contamination during sample collection may complicate downstream analyses. After discovering maternal contamination of cord blood in a cohort study of 150 neonates using Illumina 450K DNA methylation (DNAm) data, we used a combination of linear regression and random forest machine learning to create a DNAm-based screening method. We identified a panel of DNAm sites that could discriminate between contaminated and non-contaminated samples, then designed pyrosequencing assays to pre-screen DNA prior to being assayed on an array. RESULTS: Maternal contamination of cord blood was initially identified by unusual X chromosome DNA methylation patterns in 17 males. We utilized our DNAm panel to detect contaminated male samples and a proportional amount of female samples in the same cohort. We validated our DNAm screening method on an additional 189 sample cohort using both pyrosequencing and DNAm arrays, as well as 9 publically available cord blood 450K data sets. The rate of contamination varied from 0 to 10% within these studies, likely related to collection specific methods. CONCLUSIONS: Maternal blood can contaminate cord blood during sample collection at appreciable levels across multiple studies. We have identified a panel of markers that can be used to identify this contamination, either post hoc after DNAm arrays have been completed, or in advance using a targeted technique like pyrosequencing.

DNA methylation signatures of chronic alcohol dependence in purified CD3+ T-cells of patients undergoing alcohol treatment.

Several studies have shown an association of alcohol dependence with DNA methylation (DNAm), suggesting that environmentally-induced changes on epigenomic variation may play an important role in alcohol dependence. In the present study, we analysed genome-wide DNAm profiles of purified CD3+ T-cells from pre- and post-treatment alcohol dependent patients, as well as closely matched healthy controls. We identified 59 differentially methylated CpG sites comparing patients prior to treatment with healthy controls and were able to confirm 8 of those sites in additional analyses for differentially methylated regions. Comparing patients before and after a 3-week alcohol treatment program we revealed another unique set of 48 differentially methylated CpG sites. Additionally, we found that the mean global DNAm was significantly lower in patients prior to treatment compared to controls, but reverted back to levels similar to controls after treatment. We validated top-ranked hits derived from the epigenome-wide analysis by pyrosequencing and further replicated two of them in an independent cohort and confirmed differential DNAm of HECW2 and SRPK3 in whole blood. This study is the first to show widespread DNAm variation in a disease-relevant blood cell type and implicates HECW2 and SRPK3 DNAm as promising blood-based candidates to follow up in future studies.

Social and physical environments early in development predict DNA methylation of inflammatory genes in young adulthood.

Chronic inflammation contributes to a wide range of human diseases, and environments in infancy and childhood are important determinants of inflammatory phenotypes. The underlying biological mechanisms connecting early environments with the regulation of inflammation in adulthood are not known, but epigenetic processes are plausible candidates. We tested the hypothesis that patterns of DNA methylation (DNAm) in inflammatory genes in young adulthood would be predicted by early life nutritional, microbial, and psychosocial exposures previously associated with levels of inflammation. Data come from a population-based longitudinal birth cohort study in metropolitan Cebu, the Philippines, and DNAm was characterized in whole blood samples from 494 participants (age 20-22 y). Analyses focused on probes in 114 target genes involved in the regulation of inflammation, and we identified 10 sites across nine genes where the level of DNAm was significantly predicted by the following variables: household socioeconomic status in childhood, extended absence of a parent in childhood, exposure to animal feces in infancy, birth in the dry season, or duration of exclusive breastfeeding. To evaluate the biological significance of these sites, we tested for associations with a panel of inflammatory biomarkers measured in plasma obtained at the same age as DNAm assessment. Three sites predicted elevated inflammation, and one site predicted lower inflammation, consistent with the interpretation that levels of DNAm at these sites are functionally relevant. This pattern of results points toward DNAm as a potentially important biological mechanism through which developmental environments shape inflammatory phenotypes across the life course.