Twin-based Mendelian Randomization Analyses Highlight Smoking's Effects on Blood DNA Methylation, with Putative Reverse Causation.

Epigenome-wide association studies (EWAS) aim to identify differentially methylated loci associated with complex traits and disorders. EWAS of cigarette smoking shows some of the most widespread DNA methylation (DNAm) associations in blood. However, traditional EWAS cannot differentiate between causation and confounding, leading to ambiguity in etiological interpretations. Here, we apply an integrated approach combining Mendelian Randomization and twin-based Direction-of-Causation analyses (MR-DoC) to examine causality underlying smoking-associated blood DNAm changes in the Netherlands Twin Register (N=2577). Evidence across models suggests that current smoking's causal effects on DNAm likely drive many of the previous EWAS findings, implicating functional pathways relevant to several adverse health outcomes of smoking, including hemopoiesis, cell- and neuro-development, and immune regulation. Additionally, we find evidence of potential reverse causal influences at some DNAm sites, with 17 of these sites enriched for gene regulatory functional elements in the brain. The top three sites with evidence of DNAm's effects on smoking annotate to genes involved in G protein-coupled receptor signaling (GNG7, RGS3) and innate immune response (SLC15A4), elucidating potential biological risk factors for smoking. This study highlights the utility of integrating genotypic and DNAm measures in twin cohorts to clarify the causal relationships between health behaviors and blood DNAm.

Catalyzing communities of research rigour champions.

The biomedical sciences must maintain and enhance a research culture that prioritizes rigour and transparency. The US National Institute of Neurological Disorders and Stroke convened a workshop entitled 'Catalyzing Communities of Research Rigor Champions' that brought together a diverse group of leaders in promoting research rigour and transparency (identified as 'rigour champions') to discuss strategies, barriers and resources for catalyzing technical, cultural and educational changes in the biomedical sciences. This article summarizes 2 days of panels and discussions and provides an overview of critical barriers to research rigour, perspectives behind reform initiatives and considerations for stakeholders across science. Additionally, we describe applications of network science to foster, maintain and expand cultural changes related to scientific rigour and opportunities to embed rigourous practices into didactic courses, training experiences and degree programme requirements. We hope this piece provides a primer for the wider research community on current discussions and actions and inspires individuals to build, join or expand collaborative networks within their own institutions that prioritize rigourous research practices.

Neuron enriched extracellular vesicles' MicroRNA expression profiles as a marker of early life alcohol consumption.

Alcohol consumption may impact and shape brain development through perturbed biological pathways and impaired molecular functions. We investigated the relationship between alcohol consumption rates and neuron-enriched extracellular vesicles' (EVs') microRNA (miRNA) expression to better understand the impact of alcohol use on early life brain biology. Neuron-enriched EVs' miRNA expression was measured from plasma samples collected from young people using a commercially available microarray platform while alcohol consumption was measured using the Alcohol Use Disorders Identification Test. Linear regression and network analyses were used to identify significantly differentially expressed miRNAs and to characterize the implicated biological pathways, respectively. Compared to alcohol naïve controls, young people reporting high alcohol consumption exhibited significantly higher expression of three neuron-enriched EVs' miRNAs including miR-30a-5p, miR-194-5p, and miR-339-3p, although only miR-30a-5p and miR-194-5p survived multiple test correction. The miRNA-miRNA interaction network inferred by a network inference algorithm did not detect any differentially expressed miRNAs with a high cutoff on edge scores. However, when the cutoff of the algorithm was reduced, five miRNAs were identified as interacting with miR-194-5p and miR-30a-5p. These seven miRNAs were associated with 25 biological functions; miR-194-5p was the most highly connected node and was highly correlated with the other miRNAs in this cluster. Our observed association between neuron-enriched EVs' miRNAs and alcohol consumption concurs with results from experimental animal models of alcohol use and suggests that high rates of alcohol consumption during the adolescent/young adult years may impact brain functioning and development by modulating miRNA expression.

Genome-wide analysis of hepatic DNA methylation reveals impact of epigenetic aging on xenobiotic metabolism and transport genes in an aged mouse model.

Hepatic xenobiotic metabolism and transport decline with age, while intact xenobiotic metabolism is associated with longevity. However, few studies have examined the genome-wide impact of epigenetic aging on these processes. We used reduced representation bisulfite sequencing (RRBS) to map DNA methylation changes in liver DNA from mice ages 4 and 24 months. We identified several thousand age-associated differentially methylated sites (a-DMS), many of which overlapped genes encoding Phase I and Phase II drug metabolizing enzymes, in addition to ABC and SLC classes of transporters. Notable genes harboring a-DMS were Cyp1a2, Cyp2d9, and Abcc2 that encode orthologs of the human drug metabolizing enzymes CYP1A2 and CYP2D6, and the multidrug resistance protein 2 (MRP2) transporter. Cyp2d9 hypermethylation with age was significantly associated with reduced gene expression, while Abcc2 expression was unchanged with age. Cyp1a2 lost methylation with age while, counterintuitively, its expression also reduced with age. We hypothesized that age-related dysregulation of the hepatic transcriptional machinery caused down-regulation of genes despite age-related hypomethylation. Bioinformatic analysis of hypomethylated a-DMS in our sample found them to be highly enriched for hepatic nuclear factor 4 alpha (HNF4α) binding sites. HNF4α promotes Cyp1a2 expression and is downregulated with age, which could explain the reduction in Cyp1a2 expression. Overall, our study supports the broad impact of epigenetic aging on xenobiotic metabolism and transport. Future work should evaluate the interplay between hepatic nuclear receptor function and epigenetic aging. These results may have implications for studies of longevity and healthy aging.

Study protocol for the Mindful Moms Study: A randomized controlled trial evaluating a mindful movement intervention for marginalized pregnant people experiencing depression.

More than 1 in 5 pregnant people in the United States experience depressive symptoms. Although treatments exist, many people remain under- or un-treated due to concerns about stigma, side effects, and costs of medications or psychotherapy, particularly those who are marginalized (defined as those who are minoritized, low-income, or with low-educational attainment). Further, the standard depression treatments do not address social connectedness, which is a potentially modifiable factor involved in depressive symptom etiology. This protocol presents the rationale, design, and status of the two-arm longitudinal parallel group randomized controlled trial - the Mindful Moms Study - which aims to evaluate the effects and mechanisms of a group-based mindful physical activity (yoga) intervention in marginalized pregnant people with depressive symptoms (n = 200) compared to a prenatal education control group. The primary aim is to evaluate effects of group assignment on depressive symptom severity, anxiety, and perceived stress over time from baseline to six weeks postpartum. Secondary aims include understanding the role of social connectedness as a moderator of the effects and to identify genome-wide DNA methylation patterns associated with depressive symptoms and perceived social connectedness at postpartum. A focus on adequate symptom management through non-pharmacologic, accessible therapies that address social connectedness during pregnancy in marginalized women is an urgent clinical and research priority. The successful completion of this study will provide important insights into social connectedness as a mechanism to decrease depressive symptoms in a largely understudied population. Trial registration: NCT04886856.

Neuron Enriched Exosomal MicroRNA Expression Profiles as a Marker of Early Life Alcohol Consumption.

Background: Alcohol consumption may impact and shape brain development through perturbed biological pathways and impaired molecular functions. We investigated the relationship between alcohol consumption rates and neuron-enriched exosomal microRNA (miRNA) expression to better understand the impact of alcohol use on early life brain biology. Methods: Neuron-enriched exosomal miRNA expression was measured from plasma samples collected from young people using a commercially available microarray platform while alcohol consumption was measured using the Alcohol Use Disorders Identification Test. Linear regression and network analyses were used to identify significantly differentially expressed miRNAs and to characterize the implicated biological pathways, respectively. Results: Compared to alcohol naïve controls, young people reporting high alcohol consumption exhibited significantly higher expression of four neuron-enriched exosomal miRNAs including miR-30a-5p, miR-194-5p, and miR-339-3p, although only miR-30a-5p and miR-194-5p survived multiple test correction. The miRNA-miRNA interaction network inferred by a network inference algorithm did not detect any differentially expressed miRNAs with a high cutoff on edge scores. However, when the cutoff of the algorithm was reduced, five miRNAs were identified as interacting with miR-194-5p and miR-30a-5p. These seven miRNAs were associated with 25 biological functions; miR-194-5p was the most highly connected node and was highly correlated with the other miRNAs in this cluster. Conclusions: Our observed association between neuron-enriched exosomal miRNAs and alcohol consumption concurs with results from experimental animal models of alcohol use and suggests that high rates of alcohol consumption during the adolescent/young adult years may impact brain functioning and development by modulating miRNA expression.

DNA Methylation Research in Autologous Hematopoietic Stem Cell Transplant Population.

Despite increased sophistication in DNA methylation (DNAm) measurement and methods, conducting studies in specific populations such as the hematopoietic stem cell transplant (HCT) population, presents unique challenges and study design considerations. In this article, we explain the motivation for investigating DNAm in the HCT population, highlighting important study design features and key findings in a longitudinal prospective pilot study of DNAm in 32 patients undergoing autologous HCT in Central Virginia, USA. We also discuss limitations and challenges to generating robust results. We observed that HCT does not prevent high-quality DNA from being extracted from whole blood for DNAm research and that longitudinal prospective studies that span pre- and 2-months post-HCT are feasible. Critically, we did not observe significant impacts of cancer diagnosis, time since transplant, age, or chromosomal sex on overall DNAm data dimensionality. These observations demonstrate that while extreme care is required to ensure generalizable, accurate, and interpretable results, researchers should not avoid HCT-DNAm research simply for fear that the transplant procedure or presence of a cancer diagnosis will prevent meaningful conclusions from being drawn. DNAm is an attractive biomarker that is understudied in patients undergoing HCT and needs to expand to improve precise prediction of HCT outcomes.

Genetic and Environmental Variation in Continuous Phenotypes in the ABCD Study®.

Twin studies yield valuable insights into the sources of variation, covariation and causation in human traits. The ABCD Study® (abcdstudy.org) was designed to take advantage of four universities known for their twin research, neuroimaging, population-based sampling, and expertise in genetic epidemiology so that representative twin studies could be performed. In this paper we use the twin data to: (i) provide initial estimates of heritability for the wide range of phenotypes assessed in the ABCD Study using a consistent direct variance estimation approach, assuring that both data and methodology are sound; and (ii) provide an online resource for researchers that can serve as a reference point for future behavior genetic studies of this publicly available dataset. Data were analyzed from 772 pairs of twins aged 9-10 years at study inception, with zygosity determined using genotypic data, recruited and assessed at four twin hub sites. The online tool provides twin correlations and both standardized and unstandardized estimates of additive genetic, and environmental variation for 14,500 continuously distributed phenotypic features, including: structural and functional neuroimaging, neurocognition, personality, psychopathology, substance use propensity, physical, and environmental trait variables. The estimates were obtained using an unconstrained variance approach, so they can be incorporated directly into meta-analyses without upwardly biasing aggregate estimates. The results indicated broad consistency with prior literature where available and provided novel estimates for phenotypes without prior twin studies or those assessed at different ages. Effects of site, self-identified race/ethnicity, age and sex were statistically controlled. Results from genetic modeling of all 53,172 continuous variables, including 38,672 functional MRI variables, will be accessible via the user-friendly open-access web interface we have established, and will be updated as new data are released from the ABCD Study. This paper provides an overview of the initial results from the twin study embedded within the ABCD Study, an introduction to the primary research domains in the ABCD study and twin methodology, and an evaluation of the initial findings with a focus on data quality and suitability for future behavior genetic studies using the ABCD dataset. The broad introductory material is provided in recognition of the multidisciplinary appeal of the ABCD Study. While this paper focuses on univariate analyses, we emphasize the opportunities for multivariate, developmental and causal analyses, as well as those evaluating heterogeneity by key moderators such as sex, demographic factors and genetic background.

Understanding the genetics of peripartum depression: Research challenges, strategies, and opportunities.

Peripartum depression (PD) is a common mood disorder associated with negative outcomes for mother and child. PD is an understudied disorder in psychiatric genetics, and progress characterizing its genetic architecture has been limited by a lack of disorder-specific research, heterogeneous and evolving phenotypic definitions, inadequate representation of global populations, low-powered studies, and insufficient data amenable to large meta-analyses. The increasing availability of large-scale, population-level efforts, like biobanks, have the potential to accelerate scientific discovery and translational research by leveraging clinical, molecular, and self-report data from hundreds of thousands of individuals. Although these efforts will not fully equip researchers to confront every challenge posed by systemic issues in data collection, such as the reliance on minimal phenotyping strategies, the field is in a position to learn from other successful psychiatric genetic investigations. This review summarizes the current state of PD genetics research and highlights research challenges, including the impact of phenotype depth, measurement, and definition on the replicability and interpretability of genomic research. Recommendations for advancing health equity and improving the collection, analysis, discussion, and reporting of measures for PD research are provided.

Maternal biological age assessed in early pregnancy is associated with gestational age at birth.

Maternal age is an established predictor of preterm birth independent of other recognized risk factors. The use of chronological age makes the assumption that individuals age at a similar rate. Therefore, it does not capture interindividual differences that may exist due to genetic background and environmental exposures. As a result, there is a need to identify biomarkers that more closely index the rate of cellular aging. One potential candidate is biological age (BA) estimated by the DNA methylome. This study investigated whether maternal BA, estimated in either early and/or late pregnancy, predicts gestational age at birth. BA was estimated from a genome-wide DNA methylation platform using the Horvath algorithm. Linear regression methods assessed the relationship between BA and pregnancy outcomes, including gestational age at birth and prenatal perceived stress, in a primary and replication cohort. Prenatal BA estimates from early pregnancy explained variance in gestational age at birth above and beyond the influence of other recognized preterm birth risk factors. Sensitivity analyses indicated that this signal was driven primarily by self-identified African American participants. This predictive relationship was sensitive to small variations in the BA estimation algorithm. Benefits and limitations of using BA in translational research and clinical applications for preterm birth are considered.

A Primer on DNA Methylation and Its Potential to Impact Maternal Depression Risk and Assessment During Pregnancy and the Postpartum.

Depression onset during and after pregnancy is prevalent and associated with significant implications for maternal, child, and family health. Although environmental risk factors important to the expression of pregnancy-related depression are well known, knowledge of the genetic underpinning is limited. Given the joint contribution of environmental and genetic factors to depression risk liability, DNA methylation presents itself as an ideal biomarker to investigate basic mechanisms and opportunities for translational research to care for pregnancy-related depression health outcomes. This article is an introduction to DNA methylation and its potential to serve as a marker of depression risk during pregnancy and the postpartum. This commentary discusses current clinical uses of DNA methylation-based testing and how it may be applied to perinatal depression clinical care and management.

An Update on Precision Medicine Advances In Neurodevelopmental Disorders.

Neurodevelopmental disorders, including autism spectrum disorder (ASD) and attention-deficit/hyper-activity disorder (ADHD), represent a group of conditions that manifest early in child development and produce impairments across multiple domains of functioning. Although a number of pharmacological and psychosocial treatments exist to improve the symptoms associated with these syndromes, treatment advances have lagged. The Precision Medicine Initiative was launched with the goal of revolutionizing medicine by progressing beyond the historical one-size-fits-all approach. In this review, we evaluate current research efforts to personalize treatments for ASD and ADHD. Most pharmacogenetic testing has focused on the cytochrome P450 enzyme family with a particular focus on CYP2D6 and CYP2C19, which are genes that produce an enzyme that acts as a key metabolizer of many prescribed medications. This article provides an update on the state of the field of pharmacogenetics and "therapy-genetics" in the context of ASD and ADHD, and it also encourages clinicians to follow US Food and Drug Administration recommendations regarding pharmacogenetic testing.

An epigenome-wide association study of early-onset major depression in monozygotic twins.

Major depression (MD) is a debilitating mental health condition with peak prevalence occurring early in life. Genome-wide examination of DNA methylation (DNAm) offers an attractive complement to studies of allelic risk given it can reflect the combined influence of genes and environment. The current study used monozygotic twins to identify differentially and variably methylated regions of the genome that distinguish twins with and without a lifetime history of early-onset MD. The sample included 150 Caucasian monozygotic twins between the ages of 15 and 20 (73% female; Mage = 17.52 SD = 1.28) who were assessed during a developmental stage characterized by relatively distinct neurophysiological changes. All twins were generally healthy and currently free of medications with psychotropic effects. DNAm was measured in peripheral blood cells using the Infinium Human BeadChip 450 K Array. MD associations with early-onset MD were detected at 760 differentially and variably methylated probes/regions that mapped to 428 genes. Genes and genomic regions involved neural circuitry formation, projection, functioning, and plasticity. Gene enrichment analyses implicated genes related to neuron structures and neurodevelopmental processes including cell-cell adhesion genes (e.g., PCDHA genes). Genes previously implicated in mood and psychiatric disorders as well as chronic stress (e.g., NRG3) also were identified. DNAm regions associated with early-onset MD were found to overlap genetic loci identified in the latest Psychiatric Genomics Consortium meta-analysis of depression. Understanding the time course of epigenetic influences during emerging adulthood may clarify developmental phases where changes in the DNA methylome may modulate individual differences in MD risk.

Replicated umbilical cord blood DNA methylation loci associated with gestational age at birth.

DNA methylation is highly sensitive to in utero perturbations and has an established role in both embryonic development and regulation of gene expression. The foetal genetic component has been previously shown to contribute significantly to the timing of birth, yet little is known about the identity and behaviour of individual genes. The aim of this study was to test the extent genome-wide DNA methylation levels in umbilical cord blood were associated with gestational age at birth (GA). Findings were validated in an independent sample and evidence for the regulation of gene expression was evaluated for cis gene relationships in specimens with multi-omic data. Genome-wide DNA methylation, measured by the Illumina Infinium Human Methylation 450 K BeadChip, was associated with GA for 2,372 CpG probes (5% FDR) in both the Pregnancy, Race, Environment, Genes (PREG) and Newborn Epigenetic Study (NEST) cohorts. Significant probes mapped to 1,640 characterized genes and an association with nearby gene expression measures obtained by the Affymetrix HG-133A microarray was found for 11 genes. Differentially methylated positions were enriched for actively transcribed and enhancer chromatin states, were predominately located outside of CpG islands, and mapped to genes enriched for inflammation and innate immunity ontologies. In both PREG and NEST, the first principal component derived from these probes explained approximately one-half (58.1% and 47.8%, respectively) of the variation in GA. Gene pathways identified are consistent with the hypothesis of pathogen detection and response by the immune system to elicit premature labour as a consequence of unscheduled inflammation.

DNA methylation associated with postpartum depressive symptoms overlaps findings from a genome-wide association meta-analysis of depression.

BACKGROUND: Perinatal depressive symptoms have been linked to adverse maternal and infant health outcomes. The etiology associated with perinatal depressive psychopathology is poorly understood, but accumulating evidence suggests that understanding inter-individual differences in DNA methylation (DNAm) patterning may provide insight regarding the genomic regions salient to the risk liability of perinatal depressive psychopathology. RESULTS: Genome-wide DNAm was measured in maternal peripheral blood using the Infinium MethylationEPIC microarray. Ninety-two participants (46% African-American) had DNAm samples that passed all quality control metrics, and all participants were within 7 months of delivery. Linear models were constructed to identify differentially methylated sites and regions, and permutation testing was utilized to assess significance. Differentially methylated regions (DMRs) were defined as genomic regions of consistent DNAm change with at least two probes within 1 kb of each other. Maternal age, current smoking status, estimated cell-type proportions, ancestry-relevant principal components, days since delivery, and chip position served as covariates to adjust for technical and biological factors. Current postpartum depressive symptoms were measured using the Edinburgh Postnatal Depression Scale. Ninety-eight DMRs were significant (false discovery rate < 5%) and overlapped 92 genes. Three of the regions overlap loci from the latest Psychiatric Genomics Consortium meta-analysis of depression. CONCLUSIONS: Many of the genes identified in this analysis corroborate previous allelic, transcriptomic, and DNAm association results related to depressive phenotypes. Future work should integrate data from multi-omic platforms to understand the functional relevance of these DMRs and refine DNAm association results by limiting phenotypic heterogeneity and clarifying if DNAm differences relate to the timing of onset, severity, duration of perinatal mental health outcomes of the current pregnancy or to previous history of depressive psychopathology.

Heritability, stability, and prevalence of tonic and phasic irritability as indicators of disruptive mood dysregulation disorder.

BACKGROUND: Little is known about genetic and environmental influences on the components of disruptive mood dysregulation disorder (DMDD), tonic irritability (i.e., irritable mood) and phasic irritability (i.e., temper outbursts). This study examined prevalence, stability, and heritability of tonic irritability, phasic irritability, and a DMDD proxy (pDMDD) based on DSM-5 criteria. METHODS: pDMDD was derived using data from clinical interviews of parents and their twins (N = 1,431 twin pairs), ages 8-17, participating in Waves 1 and 2 of the Virginia Twin Study of Adolescent Behavioral Development. Biometrical modeling was used to compare a common pathway model (CPM) and an independent pathway model (IPM), and heritability estimates were obtained for pDMDD using the symptoms of irritable mood (tonic irritability; DMDD Criterion D), intense temper outbursts (phasic irritability; DMDD Criterion A), and frequent temper outbursts (phasic irritability; DMDD Criterion C). RESULTS: Lifetime prevalence of pDMDD was 7.46%. The stability of DMDD symptoms and the pDMDD phenotype across approximately one year were moderate (.30-.69). A CPM was a better fit to the data than an IPM. Phasic irritability loaded strongly onto the pDMDD latent factor (.89-.96) whereas tonic irritability did not (.28). Genetic influences accounted for approximately 59% of the variance in the latent pDMDD phenotype, with the remaining 41% of the variance due to unique environmental effects. The heritability of tonic irritability (54%) was slightly lower than that of frequent and intense temper (components of phasic irritability; 61% and 63%, respectively). CONCLUSIONS: Compared to tonic irritability, phasic irritability appears to be slightly more stable and heritable, as well as a stronger indicator of the latent factor. Furthermore, environmental experiences appear to play a substantial role in the development of irritability and DMDD, and researchers should seek to elucidate these mechanisms in future work.

Prospective longitudinal study of the pregnancy DNA methylome: the US Pregnancy, Race, Environment, Genes (PREG) study.

PURPOSE: The goal of the Pregnancy, Race, Environment, Genes study was to understand how social and environmental determinants of health (SEDH), pregnancy-specific environments (PSE) and biological processes influence the timing of birth and account for the racial disparity in preterm birth. The study followed a racially diverse longitudinal cohort throughout pregnancy and included repeated measures of PSE and DNA methylation (DNAm) over the course of gestation and up to 1 year into the postpartum period. PARTICIPANTS: All women were between 18 and 40 years of age with singleton pregnancies and no diagnosis of diabetes or indication of assisted reproductive technology. Both mother and father had to self-identify as either African-American (AA) or European-American (EA). Maternal peripheral blood samples along with self-report questionnaires measuring SEDH and PSE factors were collected at four pregnancy visits, and umbilical cord blood was obtained at birth. A subset of participants returned for two additional postpartum visits, during which additional questionnaires and maternal blood samples were collected. The pregnancy and postpartum extension included n=240 (AA=126; EA=114) and n=104 (AA=50; EA=54), respectively. FINDINGS TO DATE: One hundred seventy-seven women (AA=89, EA=88) met full inclusion criteria out of a total of 240 who were initially enrolled. Of the 63 participants who met exclusion criteria after enrolment, 44 (69.8%) were associated with a medical reason. Mean gestational age at birth was significantly shorter for the AA participants by 5.1 days (M=272.5 (SD=10.5) days vs M=277.6 (SD=8.3)). FUTURE PLANS: Future studies will focus on identifying key environmental factors that influence DNAm change across pregnancy and account for racial differences in preterm birth.