Autism-associated haplotype affects the regulation of the homeobox gene, ENGRAILED 2.

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 single nucleotide polymorphisms (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 = .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 linkage disequilibrium (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. Luciferase 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). The EMSAs were then performed, and nuclear factors were bound specifically to the A and C alleles of both SNPs. CONCLUSIONS: These data indicate that the A-C haplotype is functional and, together with the association and LD mapping results, supports EN2 as a likely ASD susceptibility gene and the A-C haplotype as a possible risk allele.