HGNC Approved Gene Symbol: EN2
Cytogenetic location: 7q36.3 Genomic coordinates (GRCh38): 7:155,458,129-155,464,831 (from NCBI)
In Drosophila, the 'engrailed' (en) homeobox protein plays an important role during development in segmentation, where it is required for the formation of posterior compartments (see EN1, 131290). By low stringency hybridization, Poole et al. (1989) isolated from a human cosmid genomic library sequences homologous with a probe from the Drosophila 'engrailed' gene. Partial nucleotide sequence analysis showed a consensus splice acceptor site followed by an open reading frame capable of coding 104 amino acids; the first 94 amino acids showed 71% identity with the Drosophila engrailed protein. The shared region contained a homeodomain. At the amino acid level, the human sequence was 85% identical with the mouse En1 gene and 100% identical with the mouse En2 gene.
Logan et al. (1992) isolated human and chicken genomic clones of the EN1 and EN2 genes. The deduced 333-amino acid human EN2 protein is 90% identical to mouse En1. By sequence analysis, the authors determined that En proteins from various species contain 5 distinct conserved subregions. Northern blot analysis revealed that EN2, but not EN1, is expressed as a 4-kb mRNA in human cerebellum. Similarly, Western blots indicated that only EN2 is expressed fetal cerebellum.
Logan et al. (1992) demonstrated that as in mouse and chicken, the predicted coding region of the human EN2 gene is interrupted by a single intron. Benayed et al. (2005) stated that the human EN2 gene spans 8.1 kb of genomic DNA and consists of 2 exons separated by a 3.3-kb intron.
By Southern blot analysis of DNA from a panel of human-hamster somatic cell hybrids, Poole et al. (1989) mapped the EN2 gene to human chromosome 7; regional mapping by in situ hybridization localized it to 7q36. Logan et al. (1989) arrived independently at the same assignment.
To define the function of EN2 during development, Benayed et al. (2005) expressed mouse En2 ectopically in cortical precursors. Fewer En2 transfected cells than controls displayed a differentiated phenotype. These data and results of linkage disequilibrium studies suggested that EN2 may play a role in susceptibility to autism spectrum disorder (209850).
Brunet et al. (2005) reported that an external gradient of En2 protein strongly repels growth cones of Xenopus axons originating from the temporal retina and, conversely, attracts nasal axons. Fluorescently tagged En2 accumulated inside growth cones within minutes of exposure, and a mutant form of the protein that could not enter cells failed to elicit axon turning. Once internalized, En2 stimulated the rapid phosphorylation of proteins involved in translation initiation and triggered the local synthesis of new proteins. Furthermore, the turning responses of both nasal and temporal growth cones in the presence of En2 were blocked by inhibitors of protein synthesis. Brunet et al. (2005) concluded that En2 may participate directly in topographic map formation in the vertebrate visual system.
In a study of methylation profiles in brain tissues, Ladd-Acosta et al. (2007) found that EN2 expression is decreased in brain tissue with less methylation.
Associations Pending Confirmation
Benayed et al. (2005) selected EN2 as a candidate gene for ASD (611016) because En2 mouse mutants display anatomic phenotypes in the cerebellum that are similar to those reported for individuals with autism, and because EN2 maps to a region of 7q36 that had provided suggestive evidence for linkage to ASD. Benayed et al. (2005) replicated the finding of Gharani et al. (2004) of association between ASD and 2 intronic SNPs of the EN2 gene, rs1861972 (131310.0001) and rs1861973 (131310.0002). Population-attributable risk calculations for the associated haplotype performed using their entire sample of 518 families determined that the risk allele contributes to as many as 40% of ASD cases. These data and the results of functional studies in which misexpression of mouse En2 in primary cortical cultures elicited a reduction in neuronal differentiation provided additional genetic evidence that EN2 may act as an ASD susceptibility locus, and suggested that a risk allele that perturbs the spatial/temporal expression of EN2 could significantly alter normal brain development.
To study the role of the En2 gene in development, Joyner et al. (1989) created mutations in 3 pluripotent embryonic stem cell (ES) lines by homologous recombination. Joyner et al. (1991) generated mice homozygous for a targeted deletion of the En2 homeobox. The mutant mice were viable and showed no obvious defects in embryonic development. The authors suggested that this might be due to functional redundancy of En2 and En1. They found that the mutant mice had abnormal foliation in the adult cerebellum, where normally only En2 is expressed. To determine whether the contrasting phenotypes of En2 and En1 reflect differences in temporal expression or biochemical activity of the En proteins, Hanks et al. (1995) replaced En1 coding sequences with En2 sequences in transgenic mice by gene targeting. The En2 sequences rescued all En1 mutant defects, demonstrating that the difference between En1 and En2 stems from their divergent expression patterns.
Benayed et al. (2005) stated that 2 mouse mutants exist for En2: a knockout and a transgenic that causes developmental misexpression of the gene. In both mutants, adult mice are nonataxic but the cerebellum is hypoplastic, with a decrease in the number of Purkinje cells, indicating that En2 misregulation negatively impacts cerebellar development.
Sgado et al. (2006) found that mice heterozygous null for En1 and homozygous null for En2 (En1 +/- and En2 -/-) were viable and fertile, but they showed an adult phenotype that resembled key pathologic features of Parkinson disease (168600). Mutant mice showed progressive degeneration of dopaminergic neurons in the substantia nigra, which led to diminished storage and release of dopamine in the caudate putamen, motor deficits similar to akinesia and bradykinesia, and lower body weight.
Genestine et al. (2015) found that En2 -/- mice showed dysregulated brain monoamine transmitter levels from postnatal day 7 (P7) to P21. Norepinephrine levels were reduced approximately 35% in forebrain and were increased 40 to 75% in hindbrain and cerebellum. En2 -/- forebrain regions showed reduced growth, particularly in hippocampus, where P21 dentate gyrus granule neurons were decreased 16%. Neurogenic regions of hippocampus showed increased proliferation but also highly elevated cell death, which correlated with structural changes in forebrain. En2 -/- mice had normal norepinephrine neuron numbers in brainstem. Dysregulation of monoamines was largely normalized in adult (P60) En2 -/- animals, particularly females. Adult En2 -/- males maintained reduced monoamines in frontal cortex. In addition, adult En2 -/- males, but not females, showed 2-fold higher depressive behavior, as measured by immobility in the forced swim test.
This variant, formerly titled AUTISM, ASSOCIATION WITH, 10, has been reclassified as a variant of unknown significance because its contribution to autism (611016) has not been confirmed.
In a study of 138 families in which at least 2 individuals had a strict diagnosis of autism, Gharani et al. (2004) demonstrated that a haplotype consisting of 2 intronic SNPs of the EN2 gene, the A allele of rs1861972 and the C allele of rs1861973 (131310.0002), showed significant association with autism (p = 0.000005). Less significant association (p = 0.0024) was observed for a larger group of 157 families with a broader phenotype including autism spectrum disorders. Benayed et al. (2005) replicated these association results in an additional 222 Autism Genetic Resource Exchange (AGRE) families and in 129 NIMH families.
This variant, formerly titled AUTISM, ASSOCIATION WITH, 10, has been reclassified as a variant of unknown significance because its contribution to autism (611016) has not been confirmed.
See 131310.0001, Gharani et al. (2004), and Benayed et al. (2005).
Benayed, R., Gharani, N., Rossman, I., Mancuso, V., Lazar, G., Kamdar, S., Bruse, S. E., Tischfield, S., Smith, B. J., Zimmerman, R. A., DiCicco-Bloom, E., Brzustowicz, L. M., Millonig, J. H. Support for the homeobox transcription factor gene ENGRAILED 2 as an autism spectrum disorder susceptibility locus. Am. J. Hum. Genet. 77: 851-868, 2005. [PubMed: 16252243] [Full Text: https://doi.org/10.1086/497705]
Brunet, I., Weinl, C., Piper, M., Trembleau, A., Volovitch, M., Harris, W., Prochiantz, A., Holt, C. The transcription factor Engrailed-2 guides retinal axons. Nature 438: 94-98, 2005. [PubMed: 16267555] [Full Text: https://doi.org/10.1038/nature04110]
Genestine, M., Lin, L., Durens, M., Yan, Y., Jiang, Y., Prem, S., Bailoor, K., Kelly, B., Sonsalla, P. K., Matteson, P. G., Silverman, J., Crawley, J. N., Millonig, J. H., DiCicco-Bloom, E. Engrailed-2 (En2) deletion produces multiple neurodevelopmental defects in monoamine systems, forebrain structures and neurogenesis and behavior. Hum. Molec. Genet. 24: 5805-5827, 2015. [PubMed: 26220976] [Full Text: https://doi.org/10.1093/hmg/ddv301]
Gharani, N., Benayed, R., Mancuso, V., Brzustowicz, L. M., Millonig, J. H. Association of the homeobox transcription factor, ENGRAILED 2, 3, with autism spectrum disorder. Molec. Psychiat. 9: 474-484, 2004. [PubMed: 15024396] [Full Text: https://doi.org/10.1038/sj.mp.4001498]
Hanks, M., Wurst, W., Anson-Cartwright, L., Auerbach, A. B., Joyner, A. L. Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2. Science 269: 679-682, 1995. [PubMed: 7624797] [Full Text: https://doi.org/10.1126/science.7624797]
Joyner, A. L., Herrup, K., Auerbach, B. A., Davis, C. A., Rossant, J. Subtle cerebellar phenotype in mice homozygous for a targeted deletion of the En-2 homeobox. Science 251: 1239-1243, 1991. [PubMed: 1672471] [Full Text: https://doi.org/10.1126/science.1672471]
Joyner, A. L., Skarnes, W. C., Rossant, J. Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells. Nature 338: 153-156, 1989. [PubMed: 2563902] [Full Text: https://doi.org/10.1038/338153a0]
Ladd-Acosta, C., Pevsner, J., Sabunciyan, S., Yolken, R. H., Webster, M. J., Dinkins, T., Callinan, P. A., Fan, J.-B., Potash, J. B., Feinberg, A. P. DNA methylation signatures within the human brain. Am. J. Hum. Genet. 81: 1304-1315, 2007. [PubMed: 17999367] [Full Text: https://doi.org/10.1086/524110]
Logan, C., Hanks, M. C., Noble-Topham, S., Nallainathan, D., Provart, N. J., Joyner, A. L. Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions. Dev. Genet. 13: 345-358, 1992. [PubMed: 1363401] [Full Text: https://doi.org/10.1002/dvg.1020130505]
Logan, C., Willard, H. F., Rommens, J. M., Joyner, A. L. Chromosomal localization of the human homeo box-containing genes, EN1 and EN2. Genomics 4: 206-209, 1989. [PubMed: 2567700] [Full Text: https://doi.org/10.1016/0888-7543(89)90301-7]
Poole, S. J., Law, M. L., Kao, F.-T., Lau, Y.-F. Isolation and chromosomal localization of the human En-2 gene. Genomics 4: 225-231, 1989. [PubMed: 2565873] [Full Text: https://doi.org/10.1016/0888-7543(89)90324-8]
Sgado, P., Alberi, L., Gherbassi, D., Galasso, S. L., Ramakers, G. M. J., Alavian, K. N., Smidt, M. P., Dyck, R. H., Simon, H. H. Slow progressive degeneration of nigral dopaminergic neurons in postnatal Engrailed mutant mice. Proc. Nat. Acad. Sci. 103: 15242-15247, 2006. [PubMed: 17015829] [Full Text: https://doi.org/10.1073/pnas.0602116103]