Project 1, Epidemiology & Environment, will leverage existing resources from two large epidemiologic studies to address the potential contribution from several common household exposures to risk for an autism spectrum disorder (ASD), separately and in combination with certain genomic or epigenetic profiles. The two existing investigations are: the population-based case-control CHARGE (Childhood Autism Risk from Genetics and Environment) Study, and MARBLES (Markers of Autism Risk in Babies Learning Early Signs), a cohort study following pregnant women who previously delivered a child that developed autism to understand what influences the outcome of the younger sibling and to identify early markers of ASD. Chemical classes of interest for Project 1 are: polybrominated diphenyl ethers (PBDEs, used as flame retardants), their hydroxylated metabolites, and pyrethroid insecticides (used in sprays and foggers to control ants, cockroaches, flies and mosquitos, and on pets to control fleas). Chemical determinations made in Core C in various media (plasma, urine, breastmilk) will be supplemented by toxicokinetic modeling and exposures assessed by questionnaire and other data sources. These will be examined in relation to child's developmental status, i.e., ASD, developmental delay (DD), specific speech /language delay, and other trajectories, and for associations with markers of immune function generated in Project 3, and epigenetic markers from Project 2. Differential impact of the PBDEs and pyrethroids will be evaluated based on relevant genetic polymorphisms, CNVs, or measures of global DNA methylation, considering mechanistic pathways that link to these compounds. Finally, with the bio-informatics team at Pennsylvania State University, we will explore, in discovery-oriented mode, a wide array of exposures from biologic specimens, interviews, and medical records, along with the genetic and epigenetic data.

Although autism spectrum disorder (ASD) primarily affects brain function, our data have also identified widespread changes in the immune system of children with autism (AU), both at the systemic and cellular levels. Characterization of the relationship between the immune and neuronal systems and their synergy with respect to environmental exposure is key to understanding the mechanisms through which toxicants can alter neurodevelopment. Ca2+ dependent signaling, for example through the mTOR pathway, provides a denominator that is common to both the neural and immune systems. Our data converges on specific neuro and immune-modulatory effects, implicating mTOR pathways, following ex vivo exposure of cells to congeners of PBDE (polybrominated diphenyl ethers). Project 3 hypothesizes that the maternal gestational body burden of non-coplanar environmental toxicants such as PBDE will correlate to immune dysregulation. Furthermore, we hypothesize that children with AU will exhibit increased sensitivity to PBDE exposure. We propose that increased PBDE body burden will lead to changes in the profiles of cytokines/chemokines production and that ex vivo toxicant exposure of peripheral blood mononuclear cells collected during gestation from mothers who give birth to an ASD child will be exaggerated. We further propose that children with ASD will have increased sensitivity to ex vivo exposure to PBDE leading to differentially altered immune cell function that will correlate with the altered expression of specific mTOR pathway related genes. In particular, we hypothesize that alterations in mTOR signaling will affect regulation of immune responses including changes in DNA methylation of F0XP3 expressed in regulatory T cells (Project 2). In conjunction with Project 4, we propose that there is a direct relationship between cytokine/chemokine profiles and changes in neuronal development. We have formulated these hypotheses on the basis of our published work and preliminary data demonstrating that PMBC from children with ASD respond differentially to ex vivo PBDE exposure. We will: 1) Examine the maternal gestational environment by leveraging samples taken during each trimester from mothers enrolled in the MARBLES (Markers of Autism Risk in Babies - Learning Early Signs) Study; 2) Examine the maternal gestational environment by using samples taken during each trimester from mothers enrolled in the MARBLES Study; and 3) Determine F0XP3 and global methylation on DNA from existing samples of PBDE-exposed peripheral blood mononuclear cells (CHARGE) to determine if methylation differences are reflective of differential cell function including cytokine/chemokine production.

Few rigorous epidemiologic studies have addressed the environmental causes and biologic underpinnings of autism spectrum disorder (ASD). The CHARGE (Childhood Autism Risks from Genetics and Environment) Study is a large, population-based case-control investigation of environmental risk factors, broadly defined, in relation to ASD and developmental delay without ASD symptoms, with referents from the general population. Fieldwork began in 2003, and by now CHARGE has published widely on exposures such as air pollution, mercury, flame retardants, maternal nutritional status in the peri-conception, untreated fever during pregnancy, mitochondrial dysfunction, candidate genes, a wide array of functional immune markers in both the child and the mother, and maternal metabolic conditions including obesity and diabetes. This last finding is notable, given the epidemic of obesity and type 2 diabetes that has occurred in parallel with the steady rise in ASD over the last few decades. Moreover, an emerging literature implicates several endocrine disrupting chemicals as contributing to obesity and metabolic dysregulation, including hyperinsulinemia, and to neurodevelopmental disorders as well. This project therefore builds upon these observations in several ways. First, obesity and type 2 or gestational diabetes will be examined in a larger sample, and glucose challenge test (GCT) results for the first time, to determine associations not only with development of ASD and intellectual impairment in the children, but also with specific speech and language delays, behavioral phenotypes such as attention deficits or hyperactivity, and gene expression. Second, these maternal metabolic conditions will be evaluated for associations with markers of both metabolic and immune dysregulation to be measured in neonatal bloodspots. Third, the predictive value of neonatal bloodspot markers for a later diagnosis of ASD or other child developmental and behavioral outcomes will be assessed. Fourth, the maternal metabolic conditions will be analyzed for potential links with upstream exposures to phthalates and anti- bacterial compounds, ubiquitous chemicals in common household products. Finally, this project will examine how gene variants that play a role in biochemical pathways relevant to the processes under study may influence susceptibility of the mother, the neonate or the child. To ensure adequate power for this analysis of gene-environment interaction, the sample size will be increased to 2400 children. This project addresses several goals set by the NIH Interagency Autism Coordinating Committee: 1) to discover environmental factors contributing to ASD, particularly in the prenatal period, 2) to understand biological mechanisms for ASD risk, including gene-by-environment interactions, and 3) to identify children at risk for ASD at earlier time points. Results of this detailed inquiry into early exposures, maternal pathophysiology and prognostic markers in the newborn will set the stage for developing prevention strategies.

The prevalence of autism spectrum disorders (ASDs) has been increasing during the last 10 years with increasing burden on society. While genetic risk factors are widely implicated in ASDs, the current increase is not likely to result from genetic factors alone and it is possible that environmental factors may augment known genetic risks. Current research examining biomarkers of oxidative stress and inflammation in children with an ASD suggests that these pathways may play a role in etiology. We propose to examine the association of air pollution, a common exposure that induces inflammation and oxidative stress, with autism. Using data collected as part of the Childhood Autism Risks from Genetics and the Environment Study (CHARGE) we will assess the role of traffic related air pollutants in autism risk based on monthly exposure at each child's home during prenatal, perinatal and postnatal time points. We plan to genotype 384 single nucleotide polymorphisms (SNPs) in 17 candidate genes and to examine SNP-air pollution interactions. This work may provide important insight into the etiology of ASDs and evidence for risk due to a common environmental exposure.

Autism is a severe developmental disorder characterized by deficits in social interaction and communication and by stereotypic or repetitive behaviors or restricted interests. Despite a strong influence of genetics, environmental factors are likely to play a causal role for many children. The disorder is defined purely by behavioral phenotype, with research only now burgeoning on biologic mechanisms and biochemical markers of pathogenesis. The proposed project will continue an epidemiologic study of the environmental and genetic causes of autism that was initiated under the UC Davis Center for Children's Environmental Health (CCEH) funded in the fall of 2001 by the NIEHS (1 P01 ES11269), U.S. EPA, and M.I.N.D. (Medical Investigations of Neurodevelopmental Disorders) Institute. This project, known as the CHARGE (Childhood Autism Risks from Genetics and Environment) Study, is a comprehensive, population-based case-control investigation of underlying causes for autism and triggers of regression. Cases are children with autism, and two other groups are included: children with developmental delay and children selected at random from the general population. Close to 700 participants will have been enrolled in CHARGE by the end of the first funding period of the CCEH. The proposed continuation of the CHARGE study will use the R01 mechanism due to the high cost of recruitment and data collection, which is too expensive to maintain within the CCEH, and will extend the sample size to 1600. The larger sample size will permit the examination of exposures of relatively low prevalence, of gene- environment interactions, and of etiologic specificity for subtypes of autism defined by phenotypic characteristics. The proposed study will address the possible role of exposures during the peri-conceptional, gestational, perinatal, and early childhood periods, including infections, assisted reproductive technology, medications, pesticides, brominated flame retardants, and metals. It will also evaluate these environmental factors in conjunction with genes involved in xenobiotic metabolism or suggested by our current microarray results or by SNP arrays. Finally, because autism manifests in heterogeneous phenotypes, we will examine all exposures in relation to specific subtypes of autism defined by developmental patterns such as regression versus early onset, high versus. low adaptive/cognitive function, and by immunologic profiles. This project addresses a critical and growing public health concern, namely, autism; this disorder affects as many as one in 166 children. Our project will evaluate risk factors that may play a causal role.

Recent studies indicate that immune function in children with autism spectrum disorder (ASD) is profoundly altered compared to developmentally healthy controls. There is a strong interface between the immune system and the neurologic network, and successful neurodevelopment is contingent upon a successful interaction between these two systems. We have identified several aspects of immune dysfunction in patients with autism compared with typically developing controls. These include a reduced response to vaccine antigens of bacterial origin, altered cytokine levels in plasma and upon stimulation of PBMC, increased levels of leptin in patients with early onset autism, and autoantibodies to brain antigens. This wide and complex variety of immune anomalies noted in our first funding period is in keeping with the broad range of phenotypes encompassed by the autism spectrum. Thus, we will build upon our earlier findings of both serologic and cellular changes in immune function. While our studies in the previous project period were aimed at a broad analysis of immune function in patients with autism, the current proposal will address the mechanisms responsible for the numerous alterations in immune homeostasis uncovered in our earlier studies. Therefore, our primary focus will be on the mechanisms responsible for such anomalies in immune function through an in depth analysis of cellular immune function. Our overall hypothesis is that patients with autism have a fundamental defect at the cellular level that ultimately leads to abnormalities in immune function and heightened susceptibility to environmental triggers. To examine this, we propose to: (1) examine longitudinally the serologic profile of children with ASD to ascertain whether the various immune changes noted in our first studies are maintained and/or deteriorating further; (2) determine which immune cell population(s) plays a critical role in the immune dysfunction seen in patients with autism; and (3) fully characterize the autoantibody response in a subpopulation of children with ASD and some mothers of children with ASD. It must be noted that due to the highly heterogeneous nature of autism, there will potentially be immunologic differences that relate to sub-groups of patients with autism. Therefore, we will carefully define the study groups based on our current data to include children with early onset autism, children with delayed onset/regressive autism, general population controls, and children with developmental disorders without ASD. The studies will be performed on CHARGE subjects formerly analyzed by our laboratory (CHARGE-BACK study). This will allow us to extend our prior studies longitudinally to determine if the immune dysregulation, such as increased leptin levels in the early onset patients, remains over time. The following aims address both the serologic and cellular aspects of immune function in patients with autism.