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.

Figure 1

Schematic representation of the human EN2 gene, with the location, name, and MAF of, as well as the genotyping method used for, the polymorphisms tested for association with ASD.

Figure 2

Intermarker LD values. A, Tabular representation of the pairwise LD (D′) values between the 18 polymorphisms tested for association in the AGRE I data set. The LD relationships of each polymorphism with rs1861972 and rs1861973 are highlighted, with the degree of shading corresponding to the strength of LD: black indicates strong LD (D′ > 0.72); gray indicates intermediate/weak LD (D′ range 0.024–0.632). B, The EN2 gene is illustrated with the position of all 18 tested polymorphisms demarcated by arrows. The decay of LD of each polymorphism with respect to rs1861972 and rs1861973 is represented by a gradient bar drawn below the gene. The degree of shading corresponds to the strength of LD, as described above. Only intronic polymorphisms are in strong LD with rs1861972 and rs1861973.

Figure 3

Effects of En2 misexpression on neurogenesis. A, No En2 expression in rat cerebral cortex. En2 expression was detected by RT-PCR of RNAs obtained from freshly dissected E14.5 rat cortex (Ctx) and hindbrain (HB). The primers amplified the same 220-bp region of the En2 3′ UTR from both genomic mouse (Ms) and rat DNAs, consistent with the high degree of nucleotide homology. No expression was found in rat cortex, whereas a high level was detected in the hindbrain, recapitulating the known expression pattern of En2. +/− RT = with/without reverse transcriptase; Con = control. B–D, Transfected cortical cells exhibiting morphologies of undifferentiated precursors and differentiated neurons. The arrowheads denote cells with precursor morphology, whereas the arrows identify cells with neuronal morphology. Magnification 40×; scale bar = 30 μm. B, Phase photomicrograph demonstrating cells with both precursor and neuronal morphologies. Note the large, flat cell bodies and the thick, tapering, and short processes of the precursors (arrowhead). In contrast, neuron-like cells exhibit round cell bodies with long, thin, and uniform-diameter processes (arrow). C, Examples of both precursors and neurons transfected with the En2 vectors and identified for gene expression by immunostaining for EGFP marker protein. D, Cultures that were double labeled to detect EGFP and neuron-specific cytoskeletal protein βIII-tubulin (TuJ1), an early marker of neuronal differentiation. TuJ1 expression was only detected in cells exhibiting neuronal morphologies, a finding consistent with our previous studies (Nicot and DiCicco-Bloom 2001). E, Increase in the proportion of GFP⁺ precursor cells, noted 24 h after En2 transfection. F, Corresponding decrease in the proportion of En2-transfected cells exhibiting neuronal morphology. G, En2 transfection elicited a >50% reduction in TuJ1-immunoreactive cells, a change that parallels the decrease in neuronal morphology. **P<.0005, N=9; ***P<4×10^-8, N=15.