Project description:Autism is currently considered a multigene disorder with epigenetic influences. To investigate the contribution of DNA methylation to autism spectrum disorders, we have recently completed large-scale methylation profiling by CpG island microarray analysis of lymphoblastoid cell lines (LCL) derived from monozygotic twins discordant for diagnosis of autism and their nonautistic siblings. Methylation profiling revealed many candidate genes differentially methylated between discordant MZ twins as well as between both twins and nonautistic siblings. Bioinformatics analysis of the differentially methylated genes demonstrated enrichment for high level functions including gene transcription, nervous system development, cell death/survival, and other biological processes implicated in autism. The methylation status of two of these candidate genes, BCL-2 and retinoic acid receptor (RAR)-related orphan receptor alpha (RORA), was further confirmed by bisulfite sequencing and methylation-specific PCR, respectively. Immunohistochemical analyses of tissue arrays containing slices of the cerebellum and frontal cortex of autistic and age- and sex-matched control subjects revealed decreased expression of RORA and BCL-2 proteins in the autistic brain. Our data thus confirm the role of epigenetic regulation of gene expression via differential DNA methylation in idiopathic autism, and furthermore link molecular changes in a peripheral cell model with brain pathobiology in autism. Global methylation profiling was performed on lymphoblastoid cell lines (LCLs) derived from three pairs of male monozygotic twins discordant for diagnosis of autism as determined by the Autism Diagnostic Interview-Revised (ADI-R). As controls, cell lines derived from non-autistic siblings of two pairs of twins were also included in the analyses, in addition to cell lines derived from a set of monozygotic twins unaffected by autism. For all paired analyses, a direct comparison was performed in which the methylation-enriched fractions from two individuals were pooled and hybridized onto the same microarray. In addition, indirect comparisons were performed by co-hybridizing the methylation-enriched (MIRA) fraction with the respective unenriched DNA fraction obtained from the same individual. For each paired analysis (between autistic MZ twins and/or between autistic co-twin and unaffected sibling), a total number of 4 replicates were performed, including direct and indirect comparisons.

Project description:Despite the identification of numerous autism susceptibility genes, the pathobiology of autism remains unknown. The present âcase-controlâ study takes a global approach to understanding the molecular basis of autism spectrum disorders based upon large-scale gene expression profiling. DNA microarray analyses were conducted on lymphoblastoid cell lines from over 20 sib pairs in which one sibling had a diagnosis of autism and the other was not affected in order to identify biochemical and signaling pathways which are differentially regulated in cells from autistic and nonautistic siblings. Bioinformatics and gene ontological analyses of the data implicate genes which are involved in nervous system development, inflammation, and cytoskeletal organization, in addition to genes which may be relevant to gastrointestinal or other physiological symptoms often associated with autism. Moreover, the data further suggests that these processes may be modulated by cholesterol/steroid metabolism, especially at the level of androgenic hormones. Elevation of male hormones, in turn, has been suggested as a possible factor influencing susceptibility to autism, which affects ~4 times as many males as females. Metabolic profiling of steroid hormones in lymphoblastoid cell lines from several pairs of siblings reveals higher levels of testosterone in the autistic sibling, which is consistent with the increased expression of two genes involved in the steroidogenesis pathway. Global gene expression profiling of cultured cells from ASD probands thus serves as a window to underlying metabolic and signaling deficits that may be relevant to the pathobiology of autism. Gene expression profiling of LCL from autistic (21) and nonautistic (17) siblings (4 sets of autistic twins included) were obtained using a custom-printed DNA microarray containing 39,936 elements (TIGR 40K Human array, GPL3427) and a reference design in which each sample was compared to the Stratagene Universal Human RNA standard. Following data normalization, the ratios of expression values for the autistic proband relative to his normal unaffected sibling were determined. Related siblings are identified by their common family ID# (AU****) as provided by the Autism Resource Genetic Exchange (AGRE) repository (and listed in Sample title). Differentially expressed genes were determined across all ratioed expression values for sib pairs (autistic vs. control) using one-class SAM (Statistical Analysis of Microarrays) analysis.

Project description:The expression level for 15 887 transcripts in lymphoblastoid cell lines from 19 monozygotic twin pairs (10 male, 9 female) were analysed for the effects of genotype and sex. On an average, the effect of twin pairs explained 31% of the variance in normalized gene expression levels, consistent with previous broad sense heritability estimates. The effect of sex on gene expression levels was most noticeable on the X chromosome, which contained 15 of the 20 significantly differentially expressed genes. A high concordance was observed between the sex difference test statistics and surveys of genes escaping X chromosome inactivation. Notably, several autosomal genes showed significant differences in gene expression between the sexes despite much of the cellular environment differences being effectively removed in the cell lines. A publicly available gene expression data set from the CEPH families was used to validate the results. The heritability of gene expression levels as estimated from the two data sets showed a highly significant positive correlation, particularly when both estimates were close to one and thus had the smallest standard error. There was a large concordance between the genes significantly differentially expressed between the sexes in the two data sets. Analysis of the variability of probe binding intensities within a probe set indicated that results are robust to the possible presence of polymorphisms in the target sequences. Keywords: Monozygotic twin pair Expression Profiles Genome-wide gene expression in lymphoblastoid cell lines was determined using microarrays derived from 15 monozygotic (MZ) twin pairs (10 male, 9 female). 10 twin pair are discordant, 4 are concordant for a disease phenotype and their are 5 controls which have no disease phenotype. This data set was analyzed to interrupt replicated effects of sex and genotype, not disease characteristics. Subsets of these samples have been analyzed separately for disease characteristics, (GSE7036 M-bM-^@M-^S 3 MZ twins discordant for bipolar disorder) and (GSE7486 - 5 discordant and 4 concordant MZ twin pairs with idiopathic absence epilepsies and 5 unaffected MZ twin pairs).

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