Support for the Homeobox Transcription Factor Gene ENGRAILED 2 as an Autism Spectrum Disorder Susceptibility Locus

Abstract

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.

Evidence for multiple loci from a genome scan of autism kindreds

“…This region does not overlap with the primary chromosome 7 peak from our study and from other studies (Figures 1 and 3a, b), but does include Engrailed 2 (EN2) at 180 cM, an autism candidate gene. 53 We did not find significant supporting evidence for language-related signals on other chromosomes (2, 3, 7q (B103 cM) or 13) that were identified in previous linkage studies. 13,29,30 In comparing our results to other studies, we provide strong support for a chromosome 7 autism locus, and lesser but still compelling support for loci on chromosomes 3, 4 and 11.…”

“…13,12,20 Meta-analyses of genome scan data from a subset of these studies, but not including our data, provides additional evidence for a chromosome 7q autism locus. 46,47 Autism candidate genes in the vicinity of our peak centered at B133 cM include Reelin (B112-114 cM), 48 FOXP2 (B123 cM), 49 RAY1 (B126 cM), 50 and WNT2 (B126 cM), 51 but not AUTS2 (82.16 cM) 52 or EN2 (180.84 cM) 53 (Figure 3a and b). Linkage and association studies in non-autism groups suggest that there may be a chromosome 7 gene(s) for specific language impairment 54 and dyslexia 55 at B120 and B133 cM, respectively, potentially related to autism and the signal we observe.…”

The ups and downs of Wnt signaling in prevalent neurological disorders

“…Previous studies found an association of a PvuII restriction fragment length polymorphism located in the 5 0 UTR region of EN2 in 100 autistic children and 100 control children (Petit et al, 1995). More recently, in a large number of nuclear families it was demonstrated that two intronic SNPs (SNP id: rs1861972 and rs1861973) were highly associated with the autistic phenotype, both in single locus tests as well as in haplotype analysis (Gharani et al, 2004;Benayed et al, 2005). Homozygous null mutants for En-2 in mouse display abnormal foliation patterns in the posterior half of the cerebellum and changes in Purkinje and granule cells in some posterior folia (Joyner et al, 1991;Millen et al, 1994;Vogel et al, 1996).…”

“…Currently, few studies on candidate genes which converge on linkage signals have been consistently associated with autism (Folstein and Rosen-Sheidley, 2001;Bartlett et al, 2005). Among them, and within a widely confirmed linkage region in the long arm of chromosome 7, recent evidence has shown that Wnt2 and Engrailed 2 (EN2), a Wnt/b-catenin target gene (McGrew et al, 1999), may be genetically associated with autism (Petit et al, 1995;Wassink et al, 2001;McCoy et al, 2002;Zhong et al, 2003;Gharani et al, 2004;Benayed et al, 2005).…”

“…At present, several polymorphisms in exons as well as introns and regulatory regions of the EN2 gene have been genotyped in different autistic samples including families coming from the Autism Genetic Resource Exchange (AGRE) initiative as well as to the NIMH Center for Collaborative Genetic Studies (Petit et al, 1995;Zhong et al, 2003;Gharani et al, 2004;Benayed et al, 2005). Previous studies found an association of a PvuII restriction fragment length polymorphism located in the 5 0 UTR region of EN2 in 100 autistic children and 100 control children (Petit et al, 1995).…”