Epigenetic Characterization of the FMR1 Gene and Aberrant Neurodevelopment in Human Induced Pluripotent Stem Cell Models of Fragile X Syndrome Steven D. Sheridan 1,2. , Kraig M. Theriault 1. , Surya A. Reis 1,2 , Fen Zhou 1,2 , Jon M. Madison 1,2 , Laurence Daheron 3,4 , Jeanne F. Loring 5 , Stephen J. Haggarty 1,2 * 1 Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Stanley Center for Psychiatric Research, Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 3 Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, United States of America, 4 Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 5 Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, United States of America Abstract Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. In addition to cognitive deficits, FXS patients exhibit hyperactivity, attention deficits, social difficulties, anxiety, and other autistic-like behaviors. FXS is caused by an expanded CGG trinucleotide repeat in the 59 untranslated region of the Fragile X Mental Retardation (FMR1) gene leading to epigenetic silencing and loss of expression of the Fragile X Mental Retardation protein (FMRP). Despite the known relationship between FMR1 CGG repeat expansion and FMR1 silencing, the epigenetic modifications observed at the FMR1 locus, and the consequences of the loss of FMRP on human neurodevelopment and neuronal function remain poorly understood. To address these limitations, we report on the generation of induced pluripotent stem cell (iPSC) lines from multiple patients with FXS and the characterization of their differentiation into post-mitotic neurons and glia. We show that clones from reprogrammed FXS patient fibroblast lines exhibit variation with respect to the predominant CGG-repeat length in the FMR1 gene. In two cases, iPSC clones contained predominant CGG-repeat lengths shorter than measured in corresponding input population of fibroblasts. In another instance, reprogramming a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPSC clonal lines differing in FMR1 promoter CpG methylation and FMRP expression. Using this panel of patient-specific, FXS iPSC models, we demonstrate aberrant neuronal differentiation from FXS iPSCs that is directly correlated with epigenetic modification of the FMR1 gene and a loss of FMRP expression. Overall, these findings provide evidence for a key role for FMRP early in human neurodevelopment prior to synaptogenesis and have implications for modeling of FXS using iPSC technology. By revealing disease-associated cellular phenotypes in human neurons, these iPSC models will aid in the discovery of novel therapeutics for FXS and other autism- spectrum disorders sharing common pathophysiology. Citation: Sheridan SD, Theriault KM, Reis SA, Zhou F, Madison JM, et al. (2011) Epigenetic Characterization of the FMR1 Gene and Aberrant Neurodevelopment in Human Induced Pluripotent Stem Cell Models of Fragile X Syndrome. PLoS ONE 6(10): e26203. doi:10.1371/journal.pone.0026203 Editor: Mark R. Cookson, National Institutes of Health, United States of America Received July 17, 2011; Accepted September 22, 2011; Published October 12, 2011 Copyright: ß 2011 Sheridan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by the FRAXA Research Foundation, a seed grant from the Harvard Stem Cell Institute, the Stanley Medical Research Institute, and a grant (#R33MH087896) from the National Institute Of Mental Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute Of Mental Health or the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: haggarty@chgr.mgh.harvard.edu . These authors contributed equally to this work. Introduction The autism spectrum disorders (ASDs) are a group of neuro- developmental diseases caused by multiple genetic and environ- mental factors [1]. Despite the immense etiological heterogeneity in ASDs, affected individuals have common behavioral manifes- tations that may arise due to perturbation of common neurode- velopmental processes. In the long term, identification of common cell- and molecular-level elements underlying the ASDs will require a broad study of both idiopathic and genetically correlated cases. One of the major obstacles to identification of therapeutic interventions for the ASDs has been the difficulty of studying the step-by-step development of the disease in systems that are amenable to drug and functional genomic screening. Recent advances in stem cell biology and the advent of somatic cell reprogramming technology now enable the generation of patient- specific induced pluripotent stem cells (iPSCs) that can be differentiated in vitro into a variety of cell types of the nervous system. Through the use of these patient-derived cell models, iPSCs provide a means to: i) potentially recapitulate the step-by- step development of disease, ii) discover the underlying molecular mechanisms involved in the disease pathology, and iii) apply existing and emerging approaches for discovering and testing different classes of therapeutics that target early steps in disease pathogenesis [2]. PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26203