• Quantification and Characterization of Bulk and L1CAM-Enriched Exosomal MicroRNA Cargo in Healthy Young People
    Extracellular vesicles (EVs) are membrane-bound sacs that transport bioactive materials like proteins, DNA, and RNA. EVs are released from all (or nearly all) tissues into the bloodstream as a normal part of physiology. Because EVs easily cross the blood-brain-barrier, analyzing cell surface markers and biological cargo may enable researchers to identify neuronally-derived EVs and to monitor changes in cellular functioning through differential cargo analysis. Previous work has observed that microRNAs (miRNAs) comprise a large proportion of the cargo obtained from plasma EVs. Because of their close association with genomic regulation, miRNAs represent an attractive candidate biomarker and may also be useful for identifying biological pathways associated with disease risk. A potential limitation of EV-miRNA research is that the inherent heterogeneity of EV populations may obscure mechanistic and biomarker research efforts. Attempts to reduce EV heterogeneity have produced novel protocols that extract EV subpopulations enriched for characteristics of interest like size and cell surface markers. There remains a need to evaluate the rigor, replicability, and feasibility of these extraction methods, especially in specimens from healthy young individuals. In this grant application, we will evaluate the reliability of isolating, purifying, and profiling exosomal EVs derived from blood plasma in a cohort of healthy adolescents and young adults, and we will benchmark generated outcomes. To accomplish the study aims, we will capitalize on an extant, deeply phenotyped longitudinal cohort (R01MH101518; NTotal=860; ages 15-22; 53% female) where blood plasma specimens are available for 549 participants. The cohort includes healthy adolescents/young adults (i.e., no major medical conditions) who completed a comprehensive evaluation that included measures of current/lifetime substance use (e.g., electronic/combustible cigarettes, alcohol, cannabis), psychiatric disorders, and environmental measures (e.g., life stressors/adversities, current medications). We will identify 120 plasma specimens obtained from healthy young people to accomplish three primary aims: Aim 1) Quantifying and characterizing the total number of exosomal EVs extracted from blood plasma, Aim 2) Quantifying and characterizing total RNA quantity, size representation, and RNA integrity (RIN) estimates for bulk and L1CAM-enriched exosomal EVs (Bioanalyzer) and Aim 3) Characterizing miRNA cargo present in bulk and L1CAM-enriched exosomal EV populations. Data generated by this study will inform the science of EV research in young people and provide insight into possible applications in psychopathology research.
  • Genetic, Social, and Developmental Epidemiology of Drug Use Disorders (PI: Kendler)
    This revision of a competitive renewal seeks to continue our innovative and highly productive research program which has the goal of understanding the etiology, consequences and causes of desistance of drug use disorders (DUD) utilizing data available on the entire population of Sweden of unparalleled completeness and depth. We have eight specific aims which focus on the etiology and course and consequences of DUD. These aims are: i) to develop a structural equation (SEM) based approach to co-relative analyses and then apply it to critical risk factors for DUD available in Sweden, permitting a more rigorous assessment than hitherto possible of the degree to which risk factor-DUD associations are due to familial confounding versus causal effects; ii) to examine how strongly DUD is predicted by measures of IQ and personality at age 18 available on ~ 97% of all Swedish males born 1951-1975 and to clarify the degree to which these associations are likely causal; iii) to explore the similarity and differences in risk factors for UD and smoking in over 1.2 million fertile women for whom smoking status is available as part of nurse-midwife’s reports; iv) to explore how factors related to immigration, with a focus on macro-level contextual characteristics (i.e., residential segregation), impact risk for DUD among first and second generation immigrants to Sweden using analytical Geographic Information Systems (GIS) methods; v) to clarify how risk factors for DUD vary by gender using discordant opposite-sex relative pairs and the degree to which these risk factors are specific to DUD versus shared with other key externalizing syndromes of alcohol use disorders and crime; vi) to develop a comprehensive SEM for DUD in adoptees, twins, siblings, half-siblings, and typical, not-lived-with and step- parents that will permit joint estimation of four sources of familial-environmental effects, to examine developmental dynamics in the genetic and environmental risk factors for DUD from adolescence to middle age utilizing a longitudinal twin-sibling design and to clarify key mediating mechanisms from distal to more proximal risk factors by constructing a phenotypic-developmental SEM model for DUD that incorporates a wide array of genetic and environmental risk factors; vii) to develop longitudinal models to clarify the social, psychiatric and medical consequences of DUD using co-relative designs to control for familial confounding and viii) to explore the predictors of desistance from DUD on both the latent and specified risk factor level and clarify the causal nature of these associations via co-relative designs. We will use comprehensive data from multiple nationwide data sources in Sweden on 11.8 million men and women to accomplish these goals. Applying the deep expertise of our research groups at Virginia Commonwealth and Lund University in drug abuse research, social and genetic epidemiology and causal modeling to a uniquely powerful sample, we expect this study to have important implications for DUD research, prevention and policy.
  • A Genetically Informed Study of Acute Threat Endophenotypes for Callous-Unemotional Traits (PI: Moore)
    Callous-unemotional (CU) traits predict socially debilitating outcomes including Antisocial Personality Disorder (ASPD) and violent crime in adulthood. Despite significant research, the etiology of CU traits is not well understood. The current project incorporates genetic, physiological, neuroanatomical and self-report measures to investigate the etiology of CU traits within the National Institute of Mental Health’s (NIMH) Research Domain Criteria (RDoC). Psychopathic/CU traits have long been proposed to reflect an underlying fear-deficit, and therefore this project focuses on measures corresponding with the acute fear construct within the RDoC matrix. Specifically, the physiological measures of facial eyeblink EMG in response to startle and fear-potentiated startle probes, as well as the neuroanatomical volumetric measures of regions of interest (ROIs; anterior cingulate cortex, orbitofrontal cortex, posterior cingulate cortex and amygdala) will be investigated as potential endophenotypes for CU traits. To be considered a putative endophenotype for CU traits a measure must display three characteristics: 1) association with CU traits, 2) heritability, and 3) genetic covariation with CU traits. While startle and neuroanatomy have been shown to meet the first two criteria, little to no research has investigated the third criteria. We hypothesize that startle response and neuroanatomical ROIs will meet all three criteria for endophenotypes of CU traits. Furthermore, we hypothesize that there are specific developmental periods that are most salient for the genetic and environmental factors influencing the progression of CU traits. We will address these hypotheses through the following Specific Aims: 1) investigate the phenotypic relationship between CU traits and startle, 2) examine the genetic influence on the variation and covariation of CU traits and startle throughout childhood and adolescence, and 3) investigate potential neuroanatomical ROIs associated with CU traits, and the genetic influence on the variation and covariation of ROIs and CU traits. We will accomplish these aims using a large, genetically informative, general population sample of juvenile (ages 9-20) twins (N=1696, 848 twin pairs). Biometrical structural equation modeling (i.e., `twin modeling’) will be used to decompose the variance and covariance of CU traits and potential endophenotypes proposed here into latent factors reflecting the effects of 1) additive genetics, 2) unique environment and 3) dominant genetic or shared environment. Additionally, how these influences change over the important developmental period of childhood and adolescence also will be examined. This proposal directly corresponds with the NIMH strategic plan for research, which emphasizes the need for research on 1) defining the mechanisms associated with complex behaviors and 2) determining the developmental trajectories of mental illness. The results from this study have the potential to a) confirm the use of specific measures as endophenotypes in future research on CU traits, and b) inform future developmentally-based intervention strategies.
  • Social, Developmental and Genetic Epidemiology of Alcohol Use Disorders (PI: Kendler)
    Despite the importance of both genetic and environmental contributions to alcohol use disorders (AUD), we still have limited knowledge about the dynamic and causal relationships between family, peer and neighborhood contexts and genetic risks in the development of AUD over the life course. To further our understanding of specific social and genetic effects on AUD, we propose to take a life course perspective to improve knowledge of causal mechanisms. Our specific aims are: to assess effects of the neighborhood environment, peer context and family system over the life course during sensitive developmental periods and examine the accumulated impact of neighborhood environments (using objective measures that eliminate same-source bias); to disentangle the stress (adverse environments cause AUD) vs. drift (AUD cause downward social mobility) hypotheses; to examine mediators (chains of risk) and effect modifiers in population subgroups; to distinguish family-level environment and genetic effects on AUD; and to determine the degree to which genetic risk factors moderate sensitivity to the pathogenic effects of environmental adversity. We propose to use comprehensive data from multiple nationwide data sources in Sweden. This will allow us to assess cumulative neighborhood exposures beginning in 1970 for the entire Swedish population and to conduct follow-up analyses of AUD until 2010. Our database will contain nationwide data on 11.8 million men and women whose neighborhoods of residence are geocoded and defined based on social (e.g. deprivation, crime) and physical (alcohol availability) factors. With careful ethical safeguards, national registries permit us to construct database by linking census data, family relationship data, neighborhood-level social and physical environmental records, crime data, military conscript data, cause of death records, inpatient and outpatient hospital records, and all prescription medication records. AUD diagnoses are available beginning in 1973 and individual- and neighborhood-level factors beginning in 1970. We will account for individual mobility and neighborhood change over time by using latent class growth modeling and marginal structural models. We will use propensity score matching and co- relative control designs to control for selective migration and thereby improve the ability to determine causality. Furthermore, we will produce refined assessments of neighborhood exposures from advanced GIS analytic techniques and study gene-environment interactions, which will provide a more robust basis for policy interventions and health promotion via an integrated genetics and environmental cross-disciplinary approach. Applying our expertise in human development and social and genetic epidemiology to a uniquely powerful sample, we expect this study to have important implications for AUD research, prevention and policy.
  • ABCD-USA Consortium: Twin Research Project (Site PI: Neale)
    Adolescence is a critical neurodevelopmental period associated with dramatic increases in rates of substance use. Identifying the pathways to substance use and its effects on adolescent development is critically important, as the effects of substance use during ongoing maturation likely have long-lasting effects on brain functioning and behavioral, health, and psychological outcomes. This Research Project Site application from the Twin Hub of the ABCD-USA Consortium is in response to RFA-DA-15-015; the proposal includes the University of Minnesota (hub leader), Virginia Commonwealth University, Washington University, and the University of Colorado to prospectively determine neurodevelopmental and behavioral predictors and consequences of substance use on children and adolescents. A representative community sample of 800 twin pairs, ages 9-10 years, from four sites whose researchers are leaders in twin research, SU and abuse, and neuroimaging of cognitive and emotional functioning will be tested, together with 700 singletons, contributing to the sample of 11,111 to be collected from 11 hubs across the ABCD-USA Consortium. Participants will undergo a comprehensive baseline assessment, including state-of-the-art brain imaging, comprehensive neuropsychological testing, bioassays, mobile monitoring and careful assessment of substance use, environment, psychopathological symptoms, and social functioning every 2 years. Interim annual interviews and quarterly web-based assessments will provide refined temporal resolution of behaviors, development, and life events with minimal participant burden. These Consortium-wide data obtained during the course of this project will elucidate: 1) effects of substance use patterns on the adolescent brain; 2) effects of substance use on behavioral and health outcomes; 3) bidirectional relationships between psychopathology and substance use patterns; 4) effects of individual genetic, behavioral, neurobiological, and environmental differences on risk profiles and substance use outcomes; and 5) “gateway interactions” between use of different substances. The Twin Hub proposes to use classic and co-twin control designs to study genetic vs. environmental contributions to adolescent brain/behavioral development and how these contributions predict SU propensity. Using sophisticated growth trajectory modeling techniques, we will also identify the genetic and environmentally- determined consequences of substance use on brain and behavioral development, including the assessment of gene-by-environment interactions. In addition, we will develop biospecimen resources for future studies of genomic, epigenomic, metabolomics and microbiome changes that may influence substance use and its broad health consequences. Specific to this Twin-Hub, we will obtain baseline and follow-up serum, saliva, and in some cases gut microbiota from biological samples. This work enriches the full ABCD-USA Consortium given that disentangling G and E contributions to individual risk for addiction and sensitivity to SU’s neurocognitive effects has highly significant public policy and prevention-based implications.
  • Psychometric and Genetic Assessments of Substance Use (PI: Neale)
    This project aims to develop and apply novel statistical approaches to address key challenges in the etiology of substance use, abuse and dependence. These are: high-density data; genotype by environment interplay; measurement; onset and offset; and comorbidity. Recent advances in genomic and other -omic technologies, neuroimaging, and near-continuous assessments of environment, location and behavior are creating unprecedented opportunities to understand the interplay between genetic and environmental risk factors for substance use behaviors. The project will develop new statistical models and computationally efficient software and methods to permit data mining of substance use phenotypes, genotypes and environmental measures. These models include those for: detecting GxE interaction in the presence of variable measurement precision; incorporating genetic marker and other high-density data into structural equation models to distinguish direct from indirect effects; models for gene-environment interplay, especially niche-selection; mixture distribution models of comorbidity; alternative models for symptoms co-occurrence; and regime switching to better characterize alternative pathways to outcomes. The new methods and models will be applied to datasets including measured genotypes and environmental risk factors collected in the United States, The Netherlands and Australia by project team members, The new models and methods will be disseminated freely and greatly increase the value of existing datasets and those being assembled with novel technologies.
  • Genome-Wide Gene Expression Profiling of Early-Onset Major Depression (PI: Roberson-Nay)
    Major depressive disorder (MDD) is a highly prevalent psychiatric condition associated with significant psychosocial sequelae, particularly the early-onset form. Recent advances in the field of epigenetics (i.e., environmentally-induced changes in DNA that affect gene function but not structure) are beginning to shed light on how exposure to stressful life events affords risk on a biologic level for psychiatric phenotypes such as early-onset MDD (EOMDD). Recent data suggests that one possible pathway to mood disorders is via epigenetic alterations to gene expression levels. Given the centrality of adverse life event exposure to depression, epigenetic effects have attracted interest as offering a plausible mechanism by which the environment may alter biological substrates associated with EOMDD and also increase future risk for MDD. We propose to evaluate epigenomic (i.e., genome-wide epigenetic [DNA methylation] and gene expression) mechanisms in a sample of 100 twin pairs, where 50 pairs are discordant for EOMDD, 25 pairs are concordant for EOMDD (positive controls) and 25 pairs are concordant for a negative lifetime history of EOMDD (negative controls), allowing for an unprecedented opportunity to verify epigenetic dysregulation associations that are not confounded by genetic background and/or shared familial factors. A psychiatric history measures lifetime psychiatric conditions, and a comprehensive battery assesses stressful life events, trauma exposures, personality, and stress reactivity (via cortisol) The biological endpoints that will be compared within MZ twin pairs include epigenetic (methylation) and gene expression biomarkers. The power of the discordant MZ twin design stems from its ability to control for the identical genome sequences and familial environmental risk factors shared within twin pairs, leaving only environmental/social experiences (or stochastic molecular events) unique to one member of a twin pair to account for the differential acquisition of epigenomic patterns. The MZ twin pair study design will provide a robust test of: (1) the epigenome’s cross-sectional association with EOMDD, (2) the epigenome’s ability to predict future depression risk and trajectory, and (3) whether improving depression levels are associated with a reversal of genetic biomarker profiles and vice versa.
  • Whole Genome Sequencing in Irish Multiplex Schizophrenia Families (PI: Riley)
    Although affected members of multiplex schizophrenia pedigrees have substantially elevated recurrence risk compared to singleton cases, the mean polygenic risk scores between these groups do not differ, suggesting that one source of this higher familial recurrence risk is rare, higher impact variation. We will collect whole genome sequence (WGS) from 600 affected members of multiplex schizophrenia pedigrees to identify rare variation shared by affected individuals within and between pedigrees potentially accounting for the increased recurrence risk, and reducing the `variant space’ under consideration. After QC and calling in our existing pipeline, a) familial sequence variants in the exome will be directly analyzed in 2000 Irish cases and 2000 Irish controls with 30X exome sequence data in production currently, and b) variants outside the exome will be imputed into 3600 Irish singleton schizophrenia or bipolar disorder cases and 3000 Irish population controls with GWAS framework data; 3781 additional UK10K controls with 10X WGS are available to increase analysis power. This imputed dataset will be analyzed using recently developed methods for kernel-based tests of variation aggregated over a defined interval (such as a gene) that avoid the inflation of type-1 error. We use multiple sources of genomic information to develop weights for each position in the genome (indexing the prior probability that a change at the site has functional consequence) and each variant detected (indexing the probability that observed changes have functional consequence), and we propose to improve the existing genomic information sources for this weighting in a number of ways. In aim 3, prioritized variants from aim 2a/2b will be directly genotyped in the case/control samples by custom microarray; individual genes or genesets showing enrichment of variation in cases (if any are observed) will be resequenced in the case/control sample. In Aim 4, the directly assessed genotypic and sequence data from aim 3 will be analyzed using standard methods to identify individual associated variants, and variant-enriched genes, genesets or other functional sequences. We seek to unambiguously identify 1) individual variants that are significantly more common in cases, or 2) individual genes or other functional sequences or 3) gene- or functional sequence sets enriched for variation in cases to provide critical information about the brain systems perturbed in schizophrenia, and the mechanisms by which such alleles increase risk.
  • Genome-Wide Association of Anxiety Spectrum Phenotypes: Furthering the PGC Anxiety Disorders Working Group (PI: Hettema)
    Anxiety disorders such as generalized anxiety disorder, panic disorder, and phobias are highly prevalent in the population and carry a significant burden of distress and impairment. Only a moderate proportion of patients with anxiety disorders respond adequately to current treatment interventions, and anxiety disorders are relatively understudied compared to other psychiatric syndromes. Exploring their genetic determinants will help elucidate their causes and guide research for prevention and new treatments. Research has documented roles for genetic risk factors in the development of anxiety disorders, many of which are shared with other internalizing phenotypes like depression and anxious temperament (“anxiety-spectrum”). We had conducted a preliminary genome-wide association study (GWAS) in approximately 18,000 subjects well-characterized for anxiety disorders using novel phenotypic approaches designed to capture common sources of genetic risk. We identified several genome-wide significant regions and polygenic risk variation associated with these phenotypes. The purpose of this current application is to facilitate expanded GWAS of the anxiety disorders. We will conduct GWAS in data from over 100,000 subjects, dissect the effects of shared versus disorder-specific genetic risk, and expand the phenotypes to include depressive disorders and anxious temperament. The Psychiatric Genomics Consortium (PGC) has achieved enormous progress in GWAS of psychiatric disorders, but they have not thus far focused on the primary ADs. The recent establishment of the PGC Anxiety Disorders Working Group will greatly facilitate accomplishment of the aims of this project, filling a major gap in psychiatric genetics research. This study will provide new insights into genetic mechanisms underlying the development of anxiety-spectrum disorders.
  • Cross-Species Genetic Analysis of Ethanol-Related Behavior (PI: Grotewiel)
    Alcohol-related disorders impose a substantial burden on society with far-reaching health consequences. The identification of novel genes and genetic pathways that influence alcohol-related behaviors will facilitate the development of new therapeutic interventions for alcoholism and other forms of alcohol abuse. In this project we will investigate genes and genetic pathways that have novel influences on ethanol behavior. Molecular-genetic information from this project should ultimately lead to better diagnosis, risk determination and treatment of alcohol-related disorders in humans. This project focuses on Clic4/Clic as a novel mouse/Drosophila gene that affects behavioral responses to ethanol. Preliminary studies indicate that this gene influences ethanol-related behavior in both fruit flies (Drosophila) and mice, suggesting that it has a conserved role in ethanol action. To further characterize Clic4/Clic and its associated molecular mechanisms in ethanol behavior, we have developed a coordinated study in Drosophila and mice. Using the Drosophila model, this project will identify the tissue site of Clic action (Aim 1), define the temporal requirements for Clic (Ai 2) and delineate molecular-genetic mechanisms of Clic function (Aim 3). Using the mouse model, this project will further characterize ethanol regulation of Clic4 in the brain (Aim 4A), characterize the role of mammalian Clic4 in drinking and other ethanol behaviors (Aim 4B), characterize downstream molecular responses to altered Clic4 expression (Aim 4C), and investigate the role of a focused set of molecular partners implicated in Clic4 action (Aim 4D). This project draws on the complementary expertise of two independent laboratories directed by PIs Grotewiel (fly) and Miles (mouse) and is designed to have several major points of integration. Clic4/Clic was originally implicated as a candidate gene for ethanol behavior by a series of analyses by the Miles laboratory on gene expression, linkage and association data. Subsequent genetic analysis of Clic in the fly by the Grotewiel laboratory made Clic4 a high priority locus for ethanol behavioral studies in the mouse (described as preliminary data). These studies come together to rationally support a more extensive investigation of how Clic/Clic4 influences ethanol responses in flies (Aims 1 and 2) and mice (Aims 4A and 4B). Furthermore, additional studies on ethanol-responsive genes in the mouse (Aims 4A and 4C) are now informing the design of experiments in flies that will investigate mechanisms of Clic action (Aim 3). The results of the Drosophila studies in Aim 3 will in turn guide the design of experiments in mice on mechanisms for Clic4 in mammalian ethanol behavior (Aim 4D). The deliberate cross-species integration in this project, implemented within a collaborative framework between the Miles and Grotewiel laboratories, will drive a vigorous genetic investigation of conserved mechanisms underlying ethanol behavior.
  • The Genetic and Environmental Etiology of Non-Medical Use of Prescription Drugs (PI: Gillespie)
    Non-medical use of prescription substances (NMUPS) is the use of a medication without a prescription, in ways other than prescribed, or for the experience or feelings elicited. The picture of NMUPS among young adults is bleak. Each year an estimated 2.4 million North Americans, or ~6,600 initiates per day, use prescription drugs non-medically for the first time, more than one-half are females. NMUPS is highest among young adults aged 18 to 25, with 5.3% reporting current NMUPS. NMUPS is associated with college drop out, worse employment outcomes, sexual victimization, health-jeopardizing behaviors such as driving under the influence, and high-risk sexual behavior, and psychiatric symptoms including suicide. Among adolescents, U.S. college students and young adults, it is also associated with poly-substance use including cocaine, ecstasy, and amphetamine-like substances. More than half of all drug-related emergency room visits in the U.S. can now be linked to NMUPS. NMUPS is a clear threat to public health. Deaths attributable to unintentional drug overdose of opioids and analgesic exceed those for cocaine and heroin, and continue to increase. Moreover, the death toll across all age groups from NMUPS exceeds those for all other illicit substances. Factors driving NMUPS include, but are not limited to, misperceptions about safety, increasing drug availability, along with individual risk factors and motivations. Despite the high prevalence of NMUPS, concomitant death rates, and comorbidity, the basic genetic epidemiology of NMUPS in terms of genetic and environmental risks remains unknown. Without a thorough, empirical understanding of these risk factors, prevention and intervention programs will continue to remain limited. Knowledge of these risks factors and their relation to other substances will also determine whether funding the development of NMUPS-specific diagnostic assessments is also merited. We propose secondary data analyses using two large population-based samples varying by sex, age and ethnicity to address three aims. The first is to describe the patterns of NMUP analgesics and stimulants in two population-based samples of young adults and college-aged students. We will then use basic genetic epidemiological methods to estimate the contribution of genetic and environmental risks in NMUP analgesics and stimulants. Next, advanced genetic epidemiological will be used to estimate the degree of genetic and environmental overlap between NMUP analgesics and stimulants and other licit and illicit substance use. Finally, we determine whether polygenic risk for alcohol, nicotine, cannabis, cocaine, and heroin use and substance use predicts NMUP analgesics and stimulants while examining sex, age and ethnic differences. This knowledge will significantly improve our understanding of the genetic and environmental risks behind the emerging, dangerous trends in NMUPS.
  • Research Training: Psychiatric and Statistical Genetics (PI: Neale)
    This application requests five further years of support for an Institutional National Research Service Award to cover multidisciplinary training in Psychiatric, Behavioral and Statistical Genetics. We request support for three pre-doctoral and three postdoctoral students for primary training in: i) statistical, quantitative, behavioral and molecular genetics~ ii) psychiatric nosology~ iii) neuroimaging genetics and neurobiology~ iv) clinical psychology~ iv) biostatistics. In addition to specializing in one of these areas, trainee will be exposed to all others and encouraged to study at least one other with sufficient detail to broaden their scope for future career development and interdisciplinary research. Training will usually be 4 years in duration for pre-doctoral and 2-3 years for postdoctoral students. Applications are expected from a wide variety of disciplines, including medicine, psychiatry, psychology, biostatistics, neuroscience, molecular genetics and biology. Trainees are housed in the Virginia Institute for Psychiatric and Behavioral Genetics at Virginia Commonwealth University, a modern custom-built facility with private offices, state-of-the-art computational facilities and integrated molecular genetics and experimental laboratories. Major strengths of the program include: i) broad expertise of faculty in psychiatry, psychology, genetics, neuroscience and statistics~ ii) highly productive research environment with well-funded faculty who are among the most highly cited researchers in the field~ iii) extensive experience and excellent track record of faculty in training at this level~ iv) potential for trainees to take pat in active data collection and data analysis projects~ v) access to large genetically informative datasets collected at VCU and elsewhere~ vi) direct access to genome sequencing, experimental study and neuroimaging facilities~ vii) pairing with clinical psychiatrist to attend rounds~ and viii) face-to-face instruction in responsible conduct of research from a leading author and instructor in this field.
  • Epigenetic Associations of Treatment Outcome for Posttraumatic Stress Disorder (PI: Amstadter)
    NARSAD Independent Investigator Award
  • Genetic and Environmental Risk Factors for Violent Criminal Behavior: A Swedish Twin Study (PI: Kendler)
  • Cross-Species Investigation of Gene Networks for Ethanol-Related Behaviors (PI: Kendler)
    Alcohol use disorders (AUDs) represent a major public health burden. Genetic risk factors contribute critically to susceptibility to AUDs likely a result of many variants each contributing modestly to risk. Genetic studies in animal models and humans have to date made slow progress in identifying genes individual risk variants. However, modern high-throughput approaches such as genome-wide association studies or genomic expression profiling promise to rapidly increase the pool of potential candidate genes influencing AUDs. This proposal for a P50 Alcohol Research Center presents a novel and highly integrated overall design to focus on both gene discovery and functional interpretation for the genetics of AUDs. This application is the outgrowth of a P20 Developmental Center grant that established the VCU Alcohol Research Center in 2009. Having made significant progress, we propose here to extend and enlarge that Center. Our approach includes three novel features: 1) A focus on gene networks contributing to AUD-related phenotypes, rather than single genes; 2) A cross-species genetic and genomics analysis to validate candidate genes and networks affecting ethanol behaviors; 3) A highly integrative Center design with rapid data sharing across projects through a cross-species analysis pipeline to provide ranked gene lists or networks for further experimental validation in the component projects. We request five years of support for six research projects and pilot grants for genetic studies in mice, worms, flies, and humans. Three projects will be in human genetics with a novel clinical laboratory component for assessing targeted genetic influences on human behavioral responses to ethanol in a controlled environment. All projects will be supported by an Administrative Core, an Analytic and Informatics Core and a Rodent Behavioral Core. The scientific work proposed in these projects and cores are clearly greater than the sum of their parts, due to the highly interactive structure of the VCU-ARC components. The VCU-ARC is well positioned to become a national resource making major contributions to the advancement of our understanding of the etiology of AUDs and their prevention and treatment.
  • A longitudinal methylome study to detect biomarkers predicting MDD trajectories (PI: van den Oord)

    Major depressive disorder (MDD) is a leading cause of the global disease burden with a life time prevalence of almost 15%. Genetic studies have not worked as well for MDD as for other psychiatric conditions. DNA methylation studies are a particularly promising complement. First, methylation markers may have better predictive power as methylation is directly related to gene expression. Second, methylation studies may improve disease understanding as they can account for a range of clinical disease features. For example, DNA sequence variants cannot explain the variability in age of onset or the dynamic course of MDD that is typified by exacerbations and remissions. DNA methylation studies potentially can as methylation levels show age-dependent changes and are dynamic in post-mitotic tissues in the brain. Third, the translational potential of methylation studies is profound Methylation sites are excellent modifiable targets for pharmacological interventions and as methylation is stable and can be measured cost-effectively in blood they can potentially be used in clinical settings. Our overarching goal is to identify methylation markers in existing periphera blood samples associated with clinical MDD trajectories over a six year time period. Although methylation marks in blood will not directly impact MDD, factors that affect trajectories (e.g. stress) may also affect methylation signatures in blood. As traces of these methylation changes may be preserved during cell division, indirectly our studies can also shed light on causal mechanisms. Methylation of human (non-stem cell) DNA occurs at CpG sites. As the biological knowledge is lacking to identify good candidate CpG sites, we will use next-generation sequencing to screen the >28 million CpGs in the human genome for their association with the persistence of MDD, and then replicate the top findings in independent samples using a different technology. Specifically, we will sequence 1,500 methylomes using DNA collected from the same subjects at baseline and after six years from three groups from the Netherlands Study of Depression and Anxiety: 1) controls with no MDD, 2) cases with MDD at baseline and then fully remit, and 3) cases with chronic MDD. To improve statistical power and to select the biologically most meaningful methylation markers, we will integrate other data such as genome-wide transcriptome data that is already available for these samples. Using a parallel longitudinal 3 group design, the 50 most promising sites will be replicated in 1,500 independent samples using a different technology. Successful completion of the proposed research will yield replicable methylation signatures of MDD disease trajectories with which we will start generating prediction algorithms that could eventually be used in the clinic to improve prevention, treatment, and diagnosis.

  • Developmental methylomics of childhood trauma and its health consequences (PI: van den Oord)
    By age 16, close to 2 children in 3 have suffered at least one adverse experience such as parental death, life-threatening illness, or family violence. Adversities have been robustly linked to an array of psychiatric and other medical conditions where the consequences can persist far into adulthood. The medical costs, mental health utilization, societal cost, and the psychological toll on its victims are tremendous. It is not wel understood how early adverse experiences are biologically embedded and what processes might be set into effect that would sustain long term health risks. To address these key questions we need prospective, longitudinal studies that begin in childhood and continue into adulthood and where data on adverse experiences can be linked to biosamples collected before and after adverse experiences as well as in adulthood. We propose just such a study, using already available samples from the Great Smoky Mountains Study (GSMS) and DNA methylation as the biological mechanism of interest. Methylation involves the addition of a methyl group to DNA and, in human non-embryonic cells, occurs mainly at CpGs. Animal and human research have shown that adverse events can result in persistent methylation changes with long-term phenotypic consequences. Capitalizing on these observations we propose a comprehensive study in a real life setting. First, we will use next-generation sequencing (NGS) to assay all >28 million CpGs in the human genome to study adversity-induced methylation changes and their persistence over time. To avoid false positive findings caused by pre-existing “case-control” differences (e.g. personality related or environmental factors such as poverty) we use a design that considers within-subject changes before and after DSM-IV extreme stressor events. Random assignment to trauma being impossible, this “natural experiment” is arguably the next best option this topic in children. Consistent with a model assuming a mediator role of methylation, we will select only the methylation sites that changed as a result of adversity for association testing with health risks. For the substantive and methodological reasons, we propose to treat maltreated children as a separate group in these analyses. Finally, we will replicate the 175 top findings in independent samples using a different and targeted technology to minimize the risk of false positives due to sampling and/or possibly technical errors. Successful completion of this project implies that we gained insight into how childhood adversities alters the methylome and what changes persist over time. We will also have identified processes associated with health risks in childhood/adulthood and found replicable methylation biomarkers associated with these risks. Methylation markers are stable and can be measured cost-effectively in blood, which is relatively easy to collect. Our findings therefore als have considerable translational potential as, for example, diagnostic “biomarkers of health risk” that could guide intervention strategies.
  • Connecting Familial and Sporadic Schizophrenia By Exome Sequencing and Population Imputation (PI: Riley)
    NARSAD Senior Investigator Award: Exome sequence 170 Irish multiplex family schizophrenia cases, combine these data with exome 80 existing multiplex family case and genome (4908 population controls) sequence data from the United Kingdom, 3) impute sequence variants into 515 remaining family members, 1606 sporadic cases and 1755 population control samples and 4) analyze data in the total sample of 685 family members, 1606 sporadic cases and 6753 population controls to identify variation increasing disease risk.
  • Early-Onset Major Depression and Genome-Wide Methylation Patterns (PI: Roberson-Nay)
    NARSAD Independent Investigator Award: A genome-wide methylation analysis via pyrosequencing methods will be performed to elucidate epigenetic processes associated with mood disordered pathways. A discordant twin design, where twins differ on early-onset major depression, will be used.
  • Pathways from Normal and Disordered Personality to Substance Use Disorders (PIs: Kendler & Gillespie)
    Chronic substance use (SU) and licit and illicit substance use disorders (SUDs) are debilitating and contribute substantially to global morbidity and mortality. Personality and personality disorders strongly influence risk of SU and SUDs, but we know little about the etiologic pathways involved. This project utilizes a unique resource to address a number of novel research questions concerning SU and SUDs. We will use state-of-the-art genetic epidemiological methods to identify causal and non-causal pathways from normal, abnormal and pathological personality to licit and illicit SU and SUDs using the longitudinal, phenotypically rich, and genetically informative population-based Norwegian Twin Registry (NTR). We will link the NTR data to national, population-based and clinical registries, using Norway’s unique personal identification number to study the risk factors, causes and disease mechanism as well as consequences of licit and illicit SU and SUDs. This will enable validation and identification of unreported SUD cases. The registry data will also provide objective information on demographics, socioeconomic status and psychosocial dysfunction as measured by sick leave, disability, and illness thereby enabling us to determine the causal and non-causal pathways from SU and SUD to psychosocial dysfunction. Our project has 6 scientific and one operational aim. We will use cross-sectional and longitudinal data to estimate the nature of the phenotypic associations between disinhibitory normal, abnormal and pathological personality types, SU and SUD, and correlated Axis I disorders before decomposing the associations into genetic and environmental pathways. Using complementary approaches we will model direction of causation between our key variables, while testing meditational models to clarify the role of SU as a gateway to SUDs and correlated externalizing spectrum disorders. Using genetically informative longitudinal models we will explore the causal relationship between alcohol use disorders and major depression. We will also merge NTR twin data with data from Statistics Norway to examine the phenotypic and genetic pathways from SU and SUDs to objective psychosocial dysfunction as indexed by nationwide sick leave, disability and illness. Our operational aim is to ascertain a new, fourth wave of questionnaire data on lifetime SU and SUDs from the NTR twins. This will provide statistical power to test competing longitudinal models. The modelling expertise of the collaborative group will ensure optimal exploitation of these resources. This application is submitted under a special funding mechanism “Norwegian Collaborative Projects with Research Groups in the United States” established and cofounded by the NIH and the Research Council of Norway. We propose to study how genetic and environmental risk factors in normal, abnormal and pathological personality impact and predict the risk of SU and SUDs by linking new and existing data from the NTR to national, population-based demographic and clinical registries. The same data will provide objective information on how SU and SUDs are related to sick leave, illness and disability.
  • Stress-Induced Drinking in Returning Soldiers: Genetic and Epigenetic Mechanisms (PI: Amstadter)
    The purpose of the present K02 Independent Scientist Award is threefold: first, to provide structured career development activities in psychiatric, statistical, and molecular genetics, second, to deepen my knowledge of alcohol-related phenotypes, and third, to apply these skills to the analysis of the rich genomic data yielded from my current R01, my other funded projects, as well as other data available at my institute. These goals stem directly from my funded projects and represent expansions in my current areas of expertise. Specifically, although I have the skill set needed to meet the genetic Aim of my current NIAAA R01 (AA020179 “Stress- induced Drinking in OEF/OIF Veterans: the Role of Combat History and PTSD” which is ongoing) I need increased training to fully utilize the data to ask new and deeper questions that are on the cutting edge of alcohol genetics research. Since my original training (in candidate gene designs) the field of genetics has changed significantly and increased in sophistication (e.g., genome wide association [GWAS] platforms, exom arrays, methylation arrays). These new platforms have introduced a number of analytic complexities, with multiple decision points in every stage of the process, from data cleaning to analysis to the interpretation of results. Thus, this K02 will give me the skills in the integration and harmonization of genomic data on multiple platforms (e.g., sequence, methylation, expression) to better utilize (beyond the original, now outdated aim) my NIAAA R01 data on the genomic influences on stress reactivity and subsequent alcohol use in soldiers. This aim will be achieved by hands-on training in molecular and epigenetics (including wetlab immersions), supervised statistical genetic analyses done in collaboration with leaders in the field, formal coursework and workshops, and individual tutoring by K02 consultants. Additionally, the K02 will deepen my expertise in the area of alcohol-related phenotypes, beyond stress-induced drinking, which has been the focus of my work to date. This aim will be achieved through directed readings, formal coursework, practical experience analyzing relevant datasets (e.g., NIAAA R37011408 “A Longitudinal Study of Genes, Environment and Alcohol Misuse in College Students”), and mentorship from leaders in the field (Dr. Kenneth Kendler). In summary, the overarching goal of the proposed K02 is to further my multidisciplinary independent program of research which aimed at the identification of risk and resilience factors, both biologic and psychosocial in nature, for traumatic stress related conditions (alcohol use disorders [AUDs], posttraumatic stress disorder [PTSD]) and translation of these findings into prevention and intervention programming. The K02 will provide me with protected time to fully engage in the training activities I have outlined, enabling me to coalesce my diverse training background and move my research program forward in ways not previously possible.