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Autism-Associated Haplotype Affects the Regulation of the Homeobox Gene, ENGRAILED 2

  • Rym Benayed
    Affiliations
    Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey
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  • Jiyeon Choi
    Affiliations
    Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey
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  • Paul G. Matteson
    Affiliations
    Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey
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  • Neda Gharani
    Affiliations
    Department of Genetics, Rutgers University, Piscataway, New Jersey
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  • Silky Kamdar
    Affiliations
    Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey
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  • Linda M. Brzustowicz
    Affiliations
    Department of Genetics, Rutgers University, Piscataway, New Jersey
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  • James H. Millonig
    Correspondence
    Address correspondence to James H. Millonig, Ph.D., Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854-5638
    Affiliations
    Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey

    Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey

    Department of Genetics, Rutgers University, Piscataway, New Jersey
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      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 single nucleotide polymorphisms (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 = .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 linkage disequilibrium (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. Luciferase 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). The EMSAs were then performed, and nuclear factors were bound specifically to the A and C alleles of both SNPs.

      Conclusions

      These data indicate that the A-C haplotype is functional and, together with the association and LD mapping results, supports EN2 as a likely ASD susceptibility gene and the A-C haplotype as a possible risk allele.

      Key Words

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