Laboratory of Molecular Design
NLGN4Y Gene Dosage Effects in Human and Cellular Models for Autism

      Autism Spectrum Disorders (ASD) are characterized by deficits in three core domains of behavior: reciprocal social interaction, communication, and restricted interests/stereotyped behaviors. While the etiology of ASD is multifactorial, a strong genetic component is suggested by the increased rates of autism in the siblings of probands and the greater concordance rate for ASD in monozygotic versus dizygotic twins (1). Data from large association studies suggest that gains as well as losses in copy number of the neuroligin genes can perturb synaptic function (2), and compelling evidence from human- and mouse model-based genetic studies implicates a role for synaptic molecules, including neuroligin 4, in the pathogenesis of ASD (3). One neuroligin gene, NLGN4Y, is present on the Y chromosome and may be related to the male predominance for ASD (4). A known, highly penetrant genetic risk factor for an ASD endophenotype is the presence of a supernumerary Y chromosome, as found in the male sex chromosome aneuploidy (SCA) disorder, 47,XYY syndrome (XYY). XYY occurs in ~0.1% of population-based males but has been reported in up to 1% of males with ASD (5-9). We hypothesize that increased gene dosage of NLGN4Y on the Y chromosomes in boys with XYY leads to increased gene expression, resulting in a major ASD susceptibility pathway. Our preliminary results demonstrate overexpression of NLGN4Y in lymphocytes of boys with XYY, at levels that correlated with their degree of stereotyped behaviors and restricted interests. Thus, NLGN4Y is an excellent candidate gene related to the development of an autism endophenotype in boys with XYY. This pilot proposal addresses a compelling need for characterizing the XYY-related ASD endophenotype, as well as creating agents for future imaging and quantitation of NLGN4Y expression in animal models and human subjects. These initial steps will provide tools to measure how overexpression of NLGN4Y leads to abnormal synaptic, circuit, and behavioral function in future, grant-supported, studies of transgenic NLGN4Y mouse models and boys with XYY.       Specific Aim 1. Identify ASD cognitive/behavioral endophenotypes in XYY males using standardized diagnostic tools for ASD including the Autism Diagnostic Interview (ADIR-R), the Autism Diagnostic Observation Scale-Generic (ADOS-G), and The Social Responsiveness Scale (SRS). Hypothesis: the prevalence of ASD endophenotypes will be significantly greater in boys with XYY than in the general male population. To test the hypothesis that boys with XYY manifest ASD cognitive/behavioral endophenotypes, Dr. Judith Ross (PI, TJU), Dr. Karen Gripp (co-PI, Nemours), Chief of Genetics, and Dr. Richard Fischer, (co-PI, Christiana), Pediatric Neurology, will perform the clinical evaluation and autism-related characterization of 10 subjects. We will recruit boys with XYY from the duPont Hospital for Children genetics clinic with Dr. Karen Gripp (co-PI), using the electronic medical records and will perform careful physical and behavioral evaluations. We will consider whether potential factors, including prenatal versus postnatal timing of diagnosis, socioeconomic status, and cognitive and adaptive functioning modulate the ASD-related phenotypes of boys with XYY.       Specific Aim 2: Develop cellular models for NLGN4Y overexpression, relevant to future studies in transgenic mouse models and in boys with XYY. We will also develop reagents to quantitate overexpression of neuroligin 4Y protein and NLGN4Y mRNA. We have demonstrated overexpression of NLGN4Y in lymphocytes of boys with XYY. To model this phenomenon, we will develop a tetracycline-inducible human cell line that overexpresses NLGN4Y. The cells will be used for protein staining by anti-neuroligin 4Y mAb, and NLGN4Y mRNA imaging by fluorescent or radionuclide hybridization probes, to reveal NLGN4Y expression and distribution. Thus overexpression of NLGN4Y in the cellular model will reproduce overexpression of NLGN4Y boys with XYY. We will achieve this aim in four parts: A. Dr. Diane Merry (co-PI, TJU) will develop inducible NLGN4Y-expressing HEK293 cells, circumventing limitations associated with assessing the effects of NLGN4Y overexpression directly in the human brain. B. Dr. David Colby (co-PI, UD) will prepare and validate specific neuroligin 4Y mAbs to measure overexpression in HEK293-NLGN4Y cells. C. Dr. Eric Wickstrom (co-PI, TJU) will design and synthesize NLGN4Y mRNA imaging agents to measure overexpression in HEK293-NLGN4Y cells. D. Dr. Mathew Thakur (co-PI, TJU) will test the validity of these agents in the HEK293-NLGN4Y cells, imaging and quantitating neuroligin 4Y with the new mAb, and imaging and quantitating cellular uptake of the NLGN4Y mRNA imaging agents.

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