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2003 Progress Report: Environmental Factors in the Etiology of Autism; Molecular Biomakers Core
EPA Grant Number: R829388C003Subproject: this is subproject number 003 , established and managed by the Center Director under grant R829388
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: CECEHDPR - University of California at Davis Center for the Study of Environmental Factors in the Etiology of Autism
Center Director: Pessah, Isaac N.
Title: Environmental Factors in the Etiology of Autism; Molecular Biomakers Core
Investigators: Hagerman, Paul , Denison, Michael , Gregg, Jeffrey , Rocke, David
Current Investigators: Hagerman, Paul , Gregg, Jeffrey , Sharp, Frank
Institution: University of California - Davis
EPA Project Officer: Saint, Chris
Project Period: September 30, 2001 through September 29, 2002
Project Period Covered by this Report: September 30, 2002 through September 29, 2003
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001)
Research Category: Children's Health , Health Effects
Description:
Objective:The principal objective of the Molecular Biomarkers Core is to identify patterns of altered gene expression that form a significant association with autism in human populations, or which are coupled to specific environmental factors in animal models. During this period the core has established a microarray facility that utilizes Affymetrix GeneChip technology. An essential component of the Core’s current efforts are to develop methods of analysis for microarray data in an effort to both better define, and reduce, the errors associated with the interpretation of microarray results (Geller et al 2002, in preparation).
Progress Summary:Work over the past ten months has continued to focus on microarray analysis related to human samples, and on the development of better tools for analysis of microarray data. Blood samples from Project I (CHARGE samples) are being provided to Core III in a blinded fashion, and these samples are currently being processed for RNA and banked. Periodically, blocks of coded samples will be identified for microarray analysis without revealing the status of the sample donors (autistic, cognitive delay, or control), except that specific numbers of each group are present within the block. This will allow Core III to analyze for autism-associated expression profiles without analyst bias during the data reduction process.
In addition, we have initiated and/or continued three specific projects that are designed to test both the microarray methodologies and specific features or predictions of models for autism:
Genetic/Immune model for autism – We hypothesize that there are genetic predispositions to altered immune cell activation that are manifest by differing patterns of gene expression in children with autism, relative to normally developing siblings.
Dr. Gregg and co-workers are in the process of testing the altered activation hypothesis by following the patterns of gene expression during mitogenic activation of immortalized lymphocytes using pokeweed mitogen (PWM) as a non-specific challenge and surrogate for an environmental challenge. Cell lines for three sibling pairs were obtained from the Autism Genetic Resource Exchange (AGRE). The selection criteria used in this study required that one of the two siblings be clinically diagnosed for autism using the autism clinical diagnostic procedures [for a description of the family recruitment and diagnostic assessments, see: Geshwind et al. (2001) Am J Hum Genet 69:297-302]. To provide a more rigorous assessment of the response to mitogen stimulation, microarray analysis was performed for a three-day time course following PWM stimulation.
RNA from cell lines representing three sibling pairs (n = 6) were analyzed over four time points (0,1,2,3 day), for a total of 24 arrays. Approximately 60% of the 12,330 transcripts on the U95A chips gave hybridization signals that were above background across the four time points. On the order of 400 genes were scored as differentially expressed for any given time point, but only those genes (n = 82) that showed consistent change in expression within and over time were retained for further analysis. Thus, although PWM stimulation dramatically alters expression profiles as a result of general cell activation, a much smaller fraction of genes display an autism-associated differential activation. These genes represent several different classes of proteins (e.g., MHC and immune response genes, transcription and translation regulators, etc.). A manuscript describing these pilot experiments is currently being prepared for publication.
Environmental stimulus for activation of dendritic cells – One of the central hypotheses to be tested under Project II is that environmental pressers, such as mercury compounds, will result in aberrant cell activation, particularly of cells that may be involved with the immune response.
In work performed within Project II, it has been found that extremely low levels of mercury compounds result in substantial activation of mouse dendritic cells. As an adjunct to the analysis of Project II, an analysis of the specific patterns of ethyl mercury-stimulated gene activation is being initiated in Core III as a means for defining a “normal” response to such environmental insults. Genes so identified would be used as candidates in studies of the type described above (Genetic/Immune model for autism), and to provide additional focus for studies of the CHARGE samples. One of the difficulties attending the study of expression profiles is false discovery due to the very large number of correlations being performed. Prior knowledge of specific genes whose activation accompanies various environmental insults would provide additional focus for the microarray analysis.
Identification of novel autism-associated genes – We hypothesize that the high incidence of autism associated with certain single-gene disorders is a consequence of the interplay of the altered primary gene and a small number of additional, epistatic genes, and that analysis of altered gene expression profiles in the background of the abnormal primary gene will reveal one or more of the epistatic genes.
Although autism is known to be highly heritable, the roles played by various candidate genes are not known. Moreover, there are several single-gene disorders that have a high incidence of autism (e.g., fragile X syndrome, 15qdup), suggesting that a small number of additional (epistatic) genes may give rise to autism in a background of an abnormal primary gene. Thus, the strategy of this experimental approach is to examine differences in RNA expression between autistic and non-autistic individuals, all of whom have the same primary genetic defect. In this instance, we have initiated a study of individuals who have fragile X syndrome, or who are carriers of expanded FMR1 alleles, since the incidence of autism in fragile X approaches 30% [Rogers et al. (2001) J Dev Behav Pediatr. 22:409-17].
We have thus far collected microarray data on 17 children with fragile X: four with autism, 5 with PDD-NOS, and the remainder with no autism phenotype. These data are currently being analyzed by regression analysis of several clinical measures (e.g. ADOS sub-scores) against gene expression profiles for (i) all genes represented on the U133A chip (~20,000 genes) and (ii) a subset of ~380 genes that have been implicated, directly or indirectly, in the autism phenotype. The latter approach, while more restrictive, will dramatically reduce the false discovery rate. This analysis is in progress.
Analysis of microarray data – We are continuing our process of developing an integrated approach to the analysis of microarray data. The first paper on this subject is now in press. We have developed methods and software for determination of differential expression using Affymetrix arrays for a broad variety of experimental designs: treatment vs. control, multiple treatments, and factorial designs. We have developed methods for estimation of the correct transformation scale on which to perform such analyses; the software will be ready for testing by the end of June 2003.
Geller SC, Gregg JP, Hagerman PJ, Rocke DM (2003) Transformation and Normalization of Oligonucleotide Microarray Data Bioinformatics (in press).
This work provides a method for normalization of Affymetrix GeneChips simultaneous with the determination of the transformation, producing a data set without chip or slide effects, but with constant variance and with symmetric errors.
Future Activities:The studies described above are in progress, and will continue. In addition, as the CHARGE samples (Project I) accumulate, sample blocks will be analyzed for autism-associated expression profiles.
Journal Articles:No journal articles submitted with this report: View all 4 publications for this subproject
Supplemental Keywords:Autism, GeneChip, genomics, microarray, variance stabilization, power calculations,
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ENVIRONMENTAL MANAGEMENT, Scientific Discipline, Health, RFA, PHYSICAL ASPECTS, Susceptibility/Sensitive Population/Genetic Susceptibility, Risk Assessment, Biology, Risk Assessments, Disease & Cumulative Effects, genetic susceptability, Health Risk Assessment, Physical Processes, Chemistry, Children's Health, biomarkers, exposure assessment, xenobiotics, neurological development, autism, synergistic interactions, mechanisms, human health risk, susceptibility, halogenated aromatics, etiology, gene-environment interaction, neurotoxic, biological markers, children, neurobehavioral, pesticides, chemical exposure, exposure, biomarker, neurobehavioral effects, human exposure, neurodevelopmental, neurotoxicity
Progress and Final Reports:
2002 Progress Report
Original Abstract
2004 Progress Report
2005 Progress Report
Main Center Abstract and Reports:
R829388 CECEHDPR - University of California at Davis Center for the Study of Environmental Factors in the Etiology of Autism
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829388C001 Environmental Factors in the Etiology of Autism; Analytic Biomakers (xenobiotic) Core
R829388C002 Environmental Factors in the Etiology of Autism; Cell Activation/Signaling Core
R829388C003 Environmental Factors in the Etiology of Autism; Molecular Biomakers Core
R829388C004 Environmental Factors in the Etiology of Autism; Childhood Autism Risks from Genetics and the Environment (The CHARGE Study)
R829388C005 Environmental Factors in the Etiology of Autism; Animal Models of Autism
R829388C006 Environmental Factors in the Etiology of Autism; Molecular and Cellular Mechanisms of Autism