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Discordant Monozygotic Twins as a Model for Genetic Environmental Interaction in Autism

Environmental factors appear to play an important role in the pathogenesis and evolution of autism. One of the possible mechanism by which the environment could influence autism is through epigenetic changes. Epigenetics is the study of information heritable during cell division other than the primary DNA sequence. Epigenetic programming can lead to widespread disruption of phenotypic plasticity, including in the nervous system. DNA methylation (DNAm) is a covalent modification of DNA in which a methyl group is transferred from S-adenosylmethionine to cytosine residues at CpG dinucleotides by a family of enzymes termed DNA-methyltransferases. In general, DNAm is associated with reduced expression of the affected gene. We have developed high throughput methods (Comprehensive High-throughput Arrays for Relative Methylation, CHARM) that make feasible genome-wide analysis of DNAm with extremely high sensitivity (100%) and specificity (90%). The preliminary application of CHARM to the study of monozygotic (MZ) twins discordant for the autism phenotype indicate that it is possible to detect epigenetic differences involving genes of relevance to autism’s pathogenesis (e.g. GRIN1).

Objective/Hypothesis: We hypothesize that epigenetic abnormalities contribute to the etiology of autism, and that by studying these abnormalities (1) we could better understand autism’s clinical heterogeneity as well as (2) identify genes that may have not only epigenetic changes but also genetic (i.e., sequence) abnormalities. 

Specific Aims: Based on our preliminary work, we propose two specific aims: 

  • Aim 1. To compare gene-specific DNAm across the genome, in a pair-wise fashion, of genetically identical individuals (MZ twins) who are substantially discordant for the autism phenotype. 
  • Aim 2. To validate altered methylation in autism, identified in Aim 1, using quantitative assays on unselected samples of children with autism. 

Study Design:

  • Aim 1 will extend our pilot lymphoblast-based analyses to a cohort of 16 well-characterized pairs of MZ twins (9 discordant for autism), which we previously studied from the behavioral and neuroimaging viewpoints. In this extraordinary cohort, which will allow molecular-phenotypic correlations, we will identify abnormally methylated genes using the CHARM whole-genome gene-specific array-based methylation analysis mentioned above. CHARM combines gel fractionation of McrBC-digested (methylation-specific) DNA, to separate unmethylated and methylated fractions, with hybridization to high-density custom NimbleGen arrays. 
  • Aim 2 will confirm the relevance of the DNAm alterations discovered in MZ twins, by validating them in independent well-characterized samples from autistic patients and controls. First, we will study ~50 gene targets identified by CHARM in lymphoblastoid cell lines from the AGRE repository, using highly quantitative gene-directed DNA methylation analysis (bisulfite pyrosequencing). We will include 200 lymphoid samples predominantly from the simplex collection, reflecting the familial distribution of autism in the general population, and 200 control lines. Following this, we will examine changes in gene expression associated with epigenetic abnormalities by quantitative RT-PCR. A similar approach will be applied to a well-characterized set of brain samples from 10 affected subjects and 10 controls, which will include regions implicated in autism pathogenesis and features. Finally, we will perform DNA sequencing of the top candidate genes to identify any sequence variants that may be linked to epigenetic modification in autism.

Impact: The research program proposed here will have major implications for the genetic and environmental management of individuals with autism, with the identified epigenetic biomarkers potentially incorporated into the diagnostic work-up, including carrier detection, prenatal diagnosis, and early postnatal diagnosis. The latter will lead to the implementation of intervention strategies that are not based solely on clinical diagnosis, as at present. Since epigenetic changes are potentially reversible, discoveries made under this award could also lead to new treatment strategies for autism. These new treatments may include drugs that target epigenetic processes (e.g. histone deacetylase inhibitors), currently developed for the treatment of cancer and other disorders, and/or selective molecular pathway inhibitors targeting genes identified in this study, or tailored dietary strategies. Finally, we will relate these results to sequence analysis of the same genes, relating genotype, epigenotypes and phenotype together in novel and exciting ways, that may have considerable practical value for autism research.

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