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.

Mouse mutants of the homeobox transcription factor Engrailed2 (En2) and autistic individuals display similar cerebellar morphological abnormalities, which include hypoplasia and a decrease in the number of Purkinje cells. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] Human EN2 maps to 7q36, a chromosomal region that has demonstrated suggestive linkage to autism spectrum disorder (ASD). [20][21][22] To investigate EN2 for evidence of association with ASD, four single-nucleotide polymorphisms (SNPs) (rs3735653, rs1861972, rs1861973, rs2361689) that span the majority of the 8.0 kb gene were assessed by the transmission/disequilibrium test [23][24][25][26] . Initially, 138 triads of autistic individuals and their parents were tested. Two intronic SNPs (rs1861972 and rs1861973) demonstrated significant association with autism (rs1861972, P ¼ 0.0018; rs1861973, P ¼ 0.0003; haplotype, P ¼ 0.000005). Flanking exonic SNPs (rs3735653 and rs2361689) did not display association. This analysis was then extended to include 167 small nuclear ASD pedigrees and significant association was again only observed for rs1861972 and rs1861973 under both the narrow and broad diagnostic criteria (narrow: rs1861972 P ¼ 0.0290, rs1861973 P ¼ 0.0073, haplotype P ¼ 0.0009; broad: rs1861972 P ¼ 0.0175, rs1861973 P ¼ 0.0107, haplotype P ¼ 0.0024). These data demonstrate association between a cerebellar patterning gene and ASD, suggesting a role for EN2 as a susceptibility locus and supporting a neurodevelopmental defect hypothesis in the etiology of autism.

The vacuolated lens (vl) mouse mutant causes congenital cataracts and neural tube defects (NTDs), with the NTDs being caused by abnormal neural fold apposition and fusion. Our positional cloning of vl indicates these phenotypes result from a deletion mutation in an uncharacterized orphan G protein-coupled receptor (GPCR), Gpr161. Gpr161 displays restricted expression to the lateral neural folds, developing lens, retina, limb, and CNS. Characterization of the vl mutation indicates that C-terminal tail of Gpr161 is truncated, leading to multiple effects on the protein, including reduced receptor-mediated endocytosis. We have also mapped three modifier quantitative trait loci (QTL) that affect the incidence of either the vl cataract or NTD phenotypes. Bioinformatic, sequence, genetic, and functional data have determined that Foxe3, a key regulator of lens development, is a gene responsible for the vl cataract-modifying phenotype. These studies have extended our understanding of the vl locus in three significant ways. One, the cloning of the vl locus has identified a previously uncharacterized GPCR-ligand pathway necessary for neural fold fusion and lens development, providing insight into the molecular regulation of these developmental processes. Two, our QTL analysis has established vl as a mouse model for studying the multigenic basis of NTDs and cataracts. Three, we have identified Foxe3 as a genetic modifier that interacts with Gpr161 to regulate lens development.cataracts ͉ Foxe3 ͉ spina bifida

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.

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