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Deciphering the Impact of Persistent Environmental Toxins on the Epigenetic Regulation of Autism Related Genes through Whole Genome Analysis in Sperm Open Access

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Although autism spectrum disorders (ASDs) are among the most heritable ofneurodevelopmental disorders, the rapidly rising prevalence of ASDs worldwide suggests thatenvironmental factors may interact with genetic risk for ASD. Environmental factors mayimpact the expression of genes associated with ASD through epigenetic mechanisms which, inturn, could lead to intergenerational effects influencing risk for ASDs. This study addressesenvironmentally associated epigenetic modifications in spermatocytes associated with different levels of exposure to a class of ubiquitous endocrine disrupting compounds (EDCs), the organochlorines. EDCs are of particular interest with respect to risk for autism because of their ability to interfere with hormonal signaling, which has been implicated in the regulation of RORA, a gene that our laboratory has previously shown to be differentially methylated in ASD and a master regulator of many other ASD risk genes.In this study we examine DNA methylation patterns in sperm from men in the Faroe Islands (Denmark) with higher than average exposure to environmental organochlorines as a result of a diet rich in pilot whale meat and blubber. Fat-soluble organochlorine compounds, with long half-lives both in the environment and bodily tissues, have been associated with methylomic modifications. Specifically, we investigated differences in DNA methylation in sperm from men with the highest (third tertile) and lowest (first tertile) exposures with respect to blood levels of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p’-DDE), a stable breakdown product of thepesticide DDT. We then performed bioinformatics analyses on the genes associated with thedifferentially methylated regions (DMRs) to identify genes and pathways that may predisposeoffspring to ASD.Sperm cells were purified from semen by density gradient centrifugation, and DNAwas extracted from lysates. Whole genome bisulfite sequencing (WGBS) was performed on a total of 52 samples divided into “discovery” (32 samples) and “validation” (20 samples) sets. Samples in each set were equally divided between those from the first and third tertiles with respect to levels of DDE exposure. A bioinformatics pipeline fine-tuned for low-coverage WGBS data ( https://github.com/ben-laufer>) was used to determine differential methylation between low- and high-exposure groups. Gene ontology and pathway analyses were performed using GREAT and Ingenuity Pathway Analysis (IPA) software, respectively.Preliminary analyses of the initial 32 samples (discovery set) revealed 674 differentially methylated regions (DMRs, permutation p ≤ 0.05) distributed across all genomic annotations. The most significant DMR was located in exon 7 of PTPRN2 (p=7.7E-05), an ADHD-associated gene whose DNA methylation status in blood has been linked to pesticide exposure and to smoking in sperm. Moreover, gene ontology analyses implicate significant localization of genes at the terminal bouton and axon terminus of nerve cells (Adj p  5.2E-06). Pathway analyses further support the inclusion of nervous system genes (ANKLE2, CNTNAP2, MBP, MYO16, PAX6, SPATA5, and TRAPPC9) in the DMRs (p=3.9E-03). Interestingly, the top developmental disorders revealed by IPA include autosomal dominant mental retardation (p=1.9E-04) and autism (Fisher’s Exact p=5.2E-04). Inclusion of all covariates resulted in a total of 894 nominally significant DMRs in the discovery set and 865 in the validation set. Both discovery and experimental datasets were significantly enriched in ASD-risk genes from the SFARI Gene database. When all 52 samples were combined in the methylation analysis, only one extended region covering the imprinted SNORD115 locus exhibited genome-wide significance for differential methylation. This locus is involved in neurological disorders such as Prader-Willi, Angelman, and 15q-dup syndrome and significantly overlaps with DMRs in sperm from fathers of children with ASD relative to those of control. Gene ontology analyses of DMR-associated genes show significant enrichment for brain development, neuron projection, central nervous system development, neurogenesis, neuron differentiation, and biological regulation. Results of this study show that elevated exposure to certain organochlorines is associated with differential genome-wide DNA methylation patterns in sperm. The DMRs are enriched for genes involved in neurological functions and developmental disorders, including ASD.

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