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 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 2 of these candidate genes, BCL-2 and retinoic acid-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.

Autism, a pervasive neurodevelopmental disorder manifested by deficits in social behavior and interpersonal communication, and by stereotyped, repetitive behaviors, is inexplicably biased towards males by a ratio of ∼4∶1, with no clear understanding of whether or how the sex hormones may play a role in autism susceptibility. Here, we show that male and female hormones differentially regulate the expression of a novel autism candidate gene, retinoic acid-related orphan receptor-alpha (RORA) in a neuronal cell line, SH-SY5Y. In addition, we demonstrate that RORA transcriptionally regulates aromatase, an enzyme that converts testosterone to estrogen. We further show that aromatase protein is significantly reduced in the frontal cortex of autistic subjects relative to sex- and age-matched controls, and is strongly correlated with RORA protein levels in the brain. These results indicate that RORA has the potential to be under both negative and positive feedback regulation by male and female hormones, respectively, through one of its transcriptional targets, aromatase, and further suggest a mechanism for introducing sex bias in autism.

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by delayed/abnormal language development, deficits in social interaction, repetitive behaviors and restricted interests. The heterogeneity in clinical presentation of ASD, likely due to different etiologies, complicates genetic/biological analyses of these disorders. DNA microarray analyses were conducted on 116 lymphoblastoid cell lines (LCL) from individuals with idiopathic autism who are divided into three phenotypic subgroups according to severity scores from the commonly used Autism Diagnostic Interview-Revised questionnaire and age-matched, nonautistic controls. Statistical analyses of gene expression data from control LCL against that of LCL from ASD probands identify genes for which expression levels are either quantitatively or qualitatively associated with phenotypic severity. Comparison of the significant differentially expressed genes from each subgroup relative to the control group reveals differentially expressed genes unique to each subgroup as well as genes in common across subgroups. Among the findings unique to the most severely affected ASD group are 15 genes that regulate circadian rhythm, which has been shown to have multiple effects on neurological as well as metabolic functions commonly dysregulated in autism. Among the genes common to all three subgroups of ASD are 20 novel genes mostly in putative noncoding regions, which appear to associate with androgen sensitivity and which may underlie the strong 4:1 bias toward affected males.

BackgroundAutism spectrum disorders (ASD) are neurodevelopmental disorders characterized by abnormalities in reciprocal social interactions and language development and/or usage, and by restricted interests and repetitive behaviors. Differential gene expression of neurologically relevant genes in lymphoblastoid cell lines from monozygotic twins discordant in diagnosis or severity of autism suggested that epigenetic factors such as DNA methylation or microRNAs (miRNAs) may be involved in ASD.MethodsGlobal miRNA expression profiling using lymphoblasts derived from these autistic twins and unaffected sibling controls was therefore performed using high-throughput miRNA microarray analysis. Selected differentially expressed miRNAs were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis, and the putative target genes of two of the confirmed miRNA were validated by knockdown and overexpression of the respective miRNAs.ResultsDifferentially expressed miRNAs were found to target genes highly involved in neurological functions and disorders in addition to genes involved in gastrointestinal diseases, circadian rhythm signaling, as well as steroid hormone metabolism and receptor signaling. Novel network analyses of the putative target genes that were inversely expressed relative to the relevant miRNA in these same samples further revealed an association with ASD and other co-morbid disorders, including muscle and gastrointestinal diseases, as well as with biological functions implicated in ASD, such as memory and synaptic plasticity. Putative gene targets (ID3 and PLK2) of two RT-PCR-confirmed brain-specific miRNAs (hsa-miR-29b and hsa-miR-219-5p) were validated by miRNA overexpression or knockdown assays, respectively. Comparisons of these mRNA and miRNA expression levels between discordant twins and between case-control sib pairs show an inverse relationship, further suggesting that ID3 and PLK2 are in vivo targets of the respective miRNA. Interestingly, the up-regulation of miR-23a and down-regulation of miR-106b in this study reflected miRNA changes previously reported in post-mortem autistic cerebellum by Abu-Elneel et al. in 2008. This finding validates these differentially expressed miRNAs in neurological tissue from a different cohort as well as supports the use of the lymphoblasts as a surrogate to study miRNA expression in ASD.ConclusionsFindings from this study strongly suggest that dysregulation of miRNA expression contributes to the observed alterations in gene expression and, in turn, may lead to the pathophysiological conditions underlying autism.

Background: The autism spectrum encompasses a set of complex multigenic developmental disorders that severely impact the development of language, non-verbal communication, and social skills, and are associated with odd, stereotyped, repetitive behavior and restricted interests. To date, diagnosis of these neurologically based disorders relies predominantly upon behavioral observations often prompted by delayed speech or aberrant behavior, and there are no known genes that can serve as definitive biomarkers for the disorders.

Heterogeneity in phenotypic presentation of ASD has been cited as one explanation for the difficulty in pinpointing specific genes involved in autism. Recent studies have attempted to reduce the "noise" in genetic and other biological data by reducing the phenotypic heterogeneity of the sample population. The current study employs multiple clustering algorithms on 123 item scores from the Autism Diagnostic Interview-Revised (ADI-R) diagnostic instrument of nearly 2000 autistic individuals to identify subgroups of autistic probands with clinically relevant behavioral phenotypes in order to isolate more homogeneous groups of subjects for gene expression analyses. Our combined cluster analyses suggest optimal division of the autistic probands into 4 phenotypic clusters based on similarity of symptom severity across the 123 selected item scores. One cluster is characterized by severe language deficits, while another exhibits milder symptoms across the domains. A third group possesses a higher frequency of savant skills while the fourth group exhibited intermediate severity across all domains. Grouping autistic individuals by multivariate cluster analysis of ADI-R scores reveals meaningful phenotypes of subgroups within the autistic spectrum which we show, in a related (accompanying) study, to be associated with distinct gene expression profiles.

Despite the identification of numerous autism susceptibility genes, the pathobiology of autism remains unknown. The present “case-control” 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. Preliminary 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.

BackgroundWe have recently identified the nuclear hormone receptor RORA (retinoic acid-related orphan receptor-alpha) as a novel candidate gene for autism spectrum disorder (ASD). Our independent cohort studies have consistently demonstrated the reduction of RORA transcript and/or protein levels in blood-derived lymphoblasts as well as in the postmortem prefrontal cortex and cerebellum of individuals with ASD. Moreover, we have also shown that RORA has the potential to be under negative and positive regulation by androgen and estrogen, respectively, suggesting the possibility that RORA may contribute to the male bias of ASD. However, little is known about transcriptional targets of this nuclear receptor, particularly in humans.MethodsHere we identify transcriptional targets of RORA in human neuronal cells on a genome-wide level using chromatin immunoprecipitation (ChIP) with an anti-RORA antibody followed by whole-genome promoter array (chip) analysis. Selected potential targets of RORA were then validated by an independent ChIP followed by quantitative PCR analysis. To further demonstrate that reduced RORA expression results in reduced transcription of RORA targets, we determined the expression levels of the selected transcriptional targets in RORA-deficient human neuronal cells, as well as in postmortem brain tissues from individuals with ASD who exhibit reduced RORA expression.ResultsThe ChIP-on-chip analysis reveals that RORA1, a major isoform of RORA protein in human brain, can be recruited to as many as 2,764 genomic locations corresponding to promoter regions of 2,544 genes across the human genome. Gene ontology analysis of this dataset of genes that are potentially directly regulated by RORA1 reveals statistically significant enrichment in biological functions negatively impacted in individuals with ASD, including neuronal differentiation, adhesion and survival, synaptogenesis, synaptic transmission and plasticity, and axonogenesis, as well as higher level functions such as development of the cortex and cerebellum, cognition, memory, and spatial learning. Independent ChIP-quantitative PCR analyses confirm binding of RORA1 to promoter regions of selected ASD-associated genes, including A2BP1, CYP19A1, ITPR1, NLGN1, and NTRK2, whose expression levels (in addition to HSD17B10) are also decreased in RORA1-repressed human neuronal cells and in prefrontal cortex tissues from individuals with ASD.ConclusionsFindings from this study indicate that RORA transcriptionally regulates A2BP1, CYP19A1, HSD17B10, ITPR1, NLGN1, and NTRK2, and strongly suggest that reduction of this sex hormone-sensitive nuclear receptor in the brain causes dysregulated expression of these ASD-relevant genes as well as their associated pathways and functions which, in turn, may contribute to the underlying pathobiology of ASD.