Risk to Autism Spectrum Disorder is likely a combination of genetic susceptibility and nongenetic environmental factors. Previous human genetic analysis has identified the ENGRAILED 2 (EN2) gene, an important regulator of CNS development, as a likely susceptibility locus. These studies demonstrated that certain EN2 genetic variants are inherited more often in individuals with ASD than unaffected siblings. Moreover, recent molecular analysis has demonstrated that these genetic variants are functional, increasing gene expression levels. EN2 levels are also increased in post-mortem samples from individuals with autism compared to unaffected controls.
Environmental factors can influence gene expression levels. These factors can regulate expression through the differential methylation of cytosine nucleotides in DNA regions called CpG islands. Interestingly, numerous CpG islands are present in the EN2 gene, suggesting that their differential methylation may affect EN2 levels.
To investigate this hypothesis, human neuronal cell lines that express EN2 will be treated with methyl donors and demethylating agents. The effect on DNA methylation and EN2 levels will then be quantified. In addition, methylation of the EN2 CpG islands will be quantified in the post-mortem samples and correlated with EN2 gene expression levels.
If our hypothesis is correct that EN2 levels are regulated by differential methylation, future experiments will focus on identifying environmental agents that could affect methylation and expression levels. Our long-term goal is to better define the molecular pathways disrupted in ASD so that environmental risk factors and possible therapeutic targets can be identified.