ENGRAILED 2 (EN2) Genetic and Functional Analysis

Abstract

Our autism research has focused on the homeobox transcription factor, ENGRAILED 2 (EN2). Prior to the advent of genome wide association and re-sequencing analysis, we selected EN2 as a candidate gene due to neuroanatomical similarities observed between individuals with autism and mouse En2 mutants. Animal studies have demonstrated that En2 is expressed throughout CNS development and regulates numerous cell biological processes implicated in ASD including connectivity, excitatory/inhibitory (E/I) circuit balance, and neurotransmitter development. The relevance of these functions to ASD etiology is discussed. Human genetic analysis by us determined that two intronic SNPs, rs1861972 and rs1861973, are significantly associated with Autism Spectrum Disorder (ASD). We observed the common haplotype (rs1861972-rs1861973 A-C) is over-transmitted to affected individuals while the rs1816972-rs1861973 G-T haplotype is over-represented in unaffected siblings. Significant results were observed in 3 datasets (518 families, 2336 individuals, P=.00000035). 6 other groups have also reported association of EN2 with ASD, suggesting that EN2 is an ASD susceptibility gene. These results are discussed. However if EN2 contributes to ASD risk, we would expect the ASD-associated A-C haplotype to segregate with a polymorphism that is functional and affects either the regulation or activity of EN2. Linkage disequilibrium mapping, re-sequencing and additional association analysis was performed, and identified the A-C haplotype as the best candidate for functional analysis. Luciferase assays conducted in primary mouse neuronal cultures demonstrated that the A-C haplotype functions as a transcriptional activator and specifically binds a protein complex. Transgenic mouse studies have demonstrated that the A-C haplotype is also functional, increasing gene expression in vivo. Finally, human post-mortem studies indicate EN2 levels are also increased in individuals with autism. Thus, the ASD-associated A-C haplotype is functional and increased EN2 levels are consistently correlated with ASD. Six significant CpG islands also flank human EN2. Preliminary studies indicate hypomethylation of these CpGs can also result in increased EN2 levels, suggesting