Our previous research involving 167 nuclear families from the Autism Genetic Resource Exchange (AGRE) demonstrated that two intronic SNPs, rs1861972 and rs1861973, in the homeodomain transcription factor gene ENGRAILED 2 (EN2) are significantly associated with autism spectrum disorder (ASD). In this study, significant replication of association for rs1861972 and rs1861973 is reported for two additional data sets: an independent set of 222 AGRE families (rs1861972-rs1861973 haplotype, P=.0016) and a separate sample of 129 National Institutes of Mental Health families (rs1861972-rs1861973 haplotype, P=.0431). Association analysis of the haplotype in the combined sample of both AGRE data sets (389 families) produced a P value of .0000033, whereas combining all three data sets (518 families) produced a P value of .00000035. Population-attributable risk calculations for the associated haplotype, performed using the entire sample of 518 families, determined that the risk allele contributes to as many as 40% of ASD cases in the general population. Linkage disequilibrium (LD) mapping with the use of polymorphisms distributed throughout the gene has shown that only intronic SNPs are in strong LD with rs1861972 and rs1861973. Resequencing and association analysis of all intronic SNPs have identified alleles associated with ASD, which makes them candidates for future functional analysis. Finally, to begin defining the function of EN2 during development, mouse En2 was ectopically expressed in cortical precursors. Fewer En2-transfected cells than controls displayed a differentiated phenotype. Together, these data provide further genetic evidence that EN2 might act as an ASD susceptibility locus, and they suggest that a risk allele that perturbs the spatial/temporal expression of EN2 could significantly alter normal brain development.

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Background-Association analysis identified the homeobox transcription factor, ENGRAILED 2 (EN2), as a possible Autism Spectrum Disorder (ASD) susceptibility gene (ASD [MIM 608636]; EN2 [MIM 131310]). The common alleles (underlined) of two intronic SNPs, rs1861972 (A/G) and rs1861973 (C/T), are over-transmitted to affected individuals both singly and as a haplotype in three separate datasets (518 families total, haplotype P=0.00000035). Methods: Further support that EN2 is a possible ASD susceptibility gene requires the identification of a risk allele, a DNA variant that is consistently associated with ASD but is also functional. To identify possible risk alleles, additional association analysis and LD mapping were performed. Candidate polymorphisms were then tested for functional differences by luciferase (luc) reporter transfections and Electrophoretic Mobility Shift Assays (EMSAs). Results: Association analysis of additional EN2 polymorphisms and LD mapping with Hapmap SNPs identified the rs1861972-rs1861973 haplotype as the most appropriate candidate to test for functional differences. Luc reporters for the two common rs1861972-rs1861973 haplotypes (A-C and G-T) were then transfected into human and rat cell lines as well as primary mouse neuronal cultures. In all cases the A-C haplotype resulted in a significant increase in luc levels (P<.005). EMSAs were then performed and nuclear factors bound specifically to the A and C alleles of both SNPs. Conclusions: These data indicate the AC haplotype is functional and together with the association and LD mapping results support EN2 as a likely ASD susceptibility gene and the A-C haplotype as a possible risk allele.

BackgroundThe homeobox transcription factor Engrailed2 (En2) has been studied extensively in neurodevelopment, particularly in the midbrain/hindbrain region and cerebellum, where it exhibits dynamic patterns of expression and regulates cell patterning and morphogenesis. Because of its roles in regulating cerebellar development and evidence of cerebellar pathology in autism spectrum disorder (ASD), we previously examined an ENGRAILED2 association and found evidence to support EN2 as a susceptibility gene, a finding replicated by several other investigators. However, its functions at the cell biological level remain undefined. In the mouse, En2 gene is expressed in granule neuron precursors (GNPs) just as they exit the cell cycle and begin to differentiate, raising the possibility that En2 may modulate these developmental processes.MethodsTo define En2 functions, we examined proliferation, differentiation and signaling pathway activation in En2 knockout (KO) and wild-type (WT) GNPs in response to a variety of extracellular growth factors and following En2 cDNA overexpression in cell culture. In vivo analyses of cerebellar GNP proliferation as well as responses to insulin-like growth factor-1 (IGF1) treatment were also conducted.ResultsProliferation markers were increased in KO GNPs in vivo and in 24-h cultures, suggesting En2 normally serves to promote cell cycle exit. Significantly, IGF1 stimulated greater DNA synthesis in KO than WT cells in culture, a finding associated with markedly increased phospho-S6 kinase activation. Similarly, there was three-fold greater DNA synthesis in the KO cerebellum in response to IGF1 in vivo. On the other hand, KO GNPs exhibited reduced neurite outgrowth and differentiation. Conversely, En2 overexpression increased cell cycle exit and promoted neuronal differentiation.ConclusionsIn aggregate, our observations suggest that the ASD-associated gene En2 promotes GNP cell cycle exit and differentiation, and modulates IGF1 activity during postnatal cerebellar development. Thus, genetic/epigenetic alterations of EN2 expression may impact proliferation, differentiation and IGF1 signaling as possible mechanisms that may contribute to ASD pathogenesis.

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