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

Center for Advanced Biotechnology and Medicine, Piscataway, NJ, 08854-5638, USA.
The American Journal of Human Genetics (Impact Factor: 10.93). 12/2005; 77(5):851-68. DOI: 10.1086/497705
Source: PubMed


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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    • "Given our findings that the absence of En2 resulted in increased GNP proliferation and decreased differentiation, it follows that overexpression should elicit opposite effects: increased GNP cell cycle exit and differentiation. Previously, we demonstrated that ectopic En2 overexpression in embryonic cortical precursors altered neurogenesis [7], and additional studies in HEK293 cells confirmed that transfection produces overexpression of En2 cDNA (not shown). Using these same vectors, we overexpressed En2 by lipid transfection of P7 GNPs. "
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    ABSTRACT: The 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. To 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. Proliferation 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. In 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.
    Molecular Autism 02/2014; 5(1):9. DOI:10.1186/2040-2392-5-9 · 5.41 Impact Factor
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    • "The homeobox-containing transcription factor engrailed-2 (En2) is crucially involved in the regionalization, patterning and neuronal differentiation of the midbrain and hindbrain [10-15]. Human studies indicated association of two intronic single-nucleotide polymorphisms (SNPs) in the human engrailed-2 (EN2) gene with ASD [16,17]. Furthermore, the ASD associated A-C haplotype markedly affected EN2 promoter activity when tested with a luciferase reporter assay in rat, mouse and human cell lines [18]. "
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    ABSTRACT: Transcriptome analysis has been used in autism spectrum disorder (ASD) to unravel common pathogenic pathways based on the assumption that distinct rare genetic variants or epigenetic modifications affect common biological pathways. To unravel recurrent ASD-related neuropathological mechanisms, we took advantage of the En2-/- mouse model and performed transcriptome profiling on cerebellar and hippocampal adult tissues. Cerebellar and hippocampal tissue samples from three En2-/- and wild type (WT) littermate mice were assessed for differential gene expression using microarray hybridization followed by RankProd analysis. To identify functional categories overrepresented in the differentially expressed genes, we used integrated gene-network analysis, gene ontology enrichment and mouse phenotype ontology analysis. Furthermore, we performed direct enrichment analysis of ASD-associated genes from the SFARI repository in our differentially expressed genes. Given the limited number of animals used in the study, we used permissive criteria and identified 842 differentially expressed genes in En2-/- cerebellum and 862 in the En2-/- hippocampus. Our functional analysis revealed that the molecular signature of En2-/- cerebellum and hippocampus shares convergent pathological pathways with ASD, including abnormal synaptic transmission, altered developmental processes and increased immune response. Furthermore, when directly compared to the repository of the SFARI database, our differentially expressed genes in the hippocampus showed enrichment of ASD-associated genes significantly higher than previously reported. qPCR was performed for representative genes to confirm relative transcript levels compared to those detected in microarrays. Despite the limited number of animals used in the study, our bioinformatic analysis indicates the En2-/- mouse is a valuable tool for investigating molecular alterations related to ASD.
    Molecular Autism 12/2013; 4(1):51. DOI:10.1186/2040-2392-4-51 · 5.41 Impact Factor
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    • "En2 has been linked to the neurodevelopmental disorder, autism , as it shares the chromosomal region 7q36.3 which is thought to be an autism susceptibility locus. There appears to be a genetic linkage in families with autistic members, but also broad similarities between the neuropathology seen in En2 knockout mice and autistic individuals, including cerebellar hypoplasia, decreased Purkinje cells and an anterior shift in the position of the amygdala in the cerebral cortex [63] [64] [65] [66] [67]. Further cohort studies, mainly in Chinese and Indian populations, support this [68] [69] [70]. "
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    ABSTRACT: Engrailed (En) is a member of the homeobox gene family, which encodes a homeodomain-containing transcription factor that is essential during early development. The only known site of normal adult Engrailed protein (EN) expression is in the nervous system, and it has been implicated in the development of both young-onset Parkinson's disease as well as autism. Over-expression of EN has been linked to tumour development in adults, particularly in breast, prostate, melanoma and ovarian cancers, and there is a growing interest in its role as a diagnostic and prognostic biomarker. It is hoped that further work may confirm associations between En expression and therapy-resistant, poor prognosis cancers, similar to that identified with other homeobox gene profiles.
    FEBS letters 02/2013; 587(6). DOI:10.1016/j.febslet.2013.01.054 · 3.17 Impact Factor
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