Impact of Endocrine Disruptors on the Sperm Methylome: A Risk Factor for Autism?

Background: Although autism spectrum disorders (ASDs) are among the most heritable of neurodevelopmental disorders, the rapidly rising prevalence of ASDs worldwide suggests that environmental factors may interact with genetic risk for ASD. Environmental factors may impact the expression of genes associated with ASD through epigenetic mechanisms which could lead to intergenerational effects influencing risk for ASDs. This study addresses environmentally associated epigenetic modifications in spermatocytes as a result 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.

Objectives: Here, we examine DNA methylation patterns in sperm from young men in the Faroe Islands (Denmark) with higher than average exposure to environmental organochlorines as a result of a diet that includes pilot whale meat and blubber. 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 the pesticide DDT. We then performed bioinformatics analyses on the genes associated with the differentially methylated regions (DMRs) to identify genes and pathways that may predispose offspring to ASD.

Methods: Sperm cells were purified from semen by density gradient centrifugation, and DNA was extracted from lysates using a Qiagen AllPrep DNA kit. Whole genome bisulfite sequencing (WGBS) was performed on 32 samples (16 samples from the first and third tertiles of DDE exposure) on the Illumina HiSeqX sequencer giving roughly 4x coverage after stringent quality control and quality analysis. 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 DDE exposure groups. Gene ontology and pathway analyses were performed using GREAT and Ingenuity Pathway Analysis (IPA) software, respectively.

Results: Preliminary analyses 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), a diabetes-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 over-representation of nervous system genes (ANKLE2, CNTNAP2, MBP, MYO16, PAX6, SPATA5, and TRAPPC9) in the DMRs (Fisher’s Exact p=3.9E-03). The top developmental disorders revealed by IPA include autosomal dominant mental retardation (Fisher’s Exact p=1.9E-04) and autism (Fisher’s Exact p=5.2E-04).

Conclusions: 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.