Alternative RNA Splicing in Autism Spectrum Disorders

Thursday, May 17, 2012
Sheraton Hall (Sheraton Centre Toronto)
10:00 AM
B. Stamova1, Y. Tian1, C. W. Nordahl2, M. D. Shen3, D. G. Amaral2 and F. R. Sharp1, (1)UC Davis, MIND Institute, Sacramento, CA, (2)Psychiatry and Behavioral Sciences, UC Davis M.I.N.D. Institute, Sacramento, CA, (3)M.I.N.D. Institute and Department of Psychiatry and Behavioral Sciences, University of California Davis Medical Center, Sacramento, CA
Background: Autism spectrum disorder (ASD) is clinically defined by communication and social impairments and repetitive behaviors. It is heritable, but genetic causes have only been identified for 20% of the cases. Genetic, environmental and gene-by-environment interactions have been proposed to be involved in ASD etiology.  RNA-level mechanisms merge effects of both genetics and environment. A major RNA –level mechanism is alternative splicing of pre-mRNA and it has been implicated in a number of diseases.

Objectives: We aimed to identify differences in alternative splicing in blood cells of 2-3.5 year old children with ASD compared to age, gender and race matched typically developing (TD) controls. In addition, we aimed to identify specific signatures of alternatively spliced isoforms in ASD children with normal size frontal lobes (NFL) and with large size frontal lobes (LFL).

Methods: The subjects were recruited through the Autism Phenome Project at the M.I.N.D. Institute. Blood was collected in PAXgene tubes. RNA was processed on Affymetrix Human Exon 1.0 expression arrays. Brain MRI and expression data was available for 30 ASD boys (age= 36.7±5.1 months), of which 20 with NFL and 10 with LFL; and 20 TD boys (age= 36.5±4.5 months). LFL was defined as a mean frontal lobe volume of > two SDs greater than the average frontal lobe volume of matched TD controls. Alternative Splicing Analysis was performed in Partek, using Splicing ANOVA (µ+Group+Exon+(Group x Exon)+ε) (p<0.005). Pathway analysis of alternatively spliced genes and differential expression at exon-level resolution was performed in IPA (adjusted p<0.05).

Results: 371 genes are predicted to be alternatively spliced in children with ASD compared to TD controls. They were over-represented in monocyte/macrophage-related pathways, such as production of nitric oxide and reactive oxygen species in macrophages and FCγ receptor-mediated phagocytosis in macrophages and monocytes, as well as in xenobiotic metabolism signaling and dendritic cell maturation. A different molecular signature was associated with ASD children based on the size of their frontal lobe. Genes associated with ASD-LFL were over expressed in IP3, apoptosis, Natural Killer cell, monocyte and TNFR1 signaling pathways. Genes associated with ASD-NFL were associated with axonal guidance and BMP receptor (TGF superfamily) pathways.

Conclusions: We provide evidence for altered alternative splicing in blood of ASD children, at a very young age, when ASD becomes evident. The majority belonged to macrophage-related pathways. The macrophage transcriptome is very similar to microglia (brain macrophage), and microglia have been shown to be activated in ASD brain (Morgan et al, 2010). Moreover, we found a significant number (45, p<1e-04) of alternatively spliced genes in ASD blood cells, which were also reported to be aberrantly spliced in ASD brain (Voineagu et al, 2011). These overlapping genes provide strong evidence that assessing blood can provide insights into ASD. This is the first study to demonstrate abnormal alternative splicing in the blood of subjects with ASD and at an early age when ASD becomes evident and requires early treatment. The findings can direct searches for environmental causes, treatment and/or prevention and can be used for biomarkers for disease diagnosis.

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