Elevated exposures to persistent endocrine disrupting compounds impact the sperm methylome in regions associated with autism spectrum disorder

Abstract

Environmental exposures to endocrine disrupting compounds (EDCs) such as the organochlorines have been linked with various diseases including neurodevelopmental disorders. Autism spectrum disorder (ASD) is a highly complex neurodevelopmental disorder that is considered strongly genetic in origin due to its high heritability. However, the rapidly rising prevalence of ASD suggests that environmental factors may also influence risk for ASD. In the present study, whole genome bisulfite sequencing was used to identify genome-wide differentially methylated regions (DMRs) in a total of 52 sperm samples from a cohort of men from the Faroe Islands (Denmark) who were equally divided into high and low exposure groups based on their serum levels of the long-lived organochlorine 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a primary breakdown product of the now banned insecticide dichlorodiphenyltrichloroethane (DDT). Aside from being considered a genetic isolate, inhabitants of the Faroe Islands have a native diet that potentially exposes them to a wide range of seafood neurotoxicants in the form of persistent organic pollutants (POPs). The DMRs were mapped to the human genome using Bismark, a 3-letter aligner used for methyl-seq analyses. Gene ontology, functional, and pathway analyses of the DMR-associated genes showed significant enrichment for genes involved in neurological functions and neurodevelopmental processes frequently impacted by ASD. Notably, these genes also significantly overlap with autism risk genes as well as those previously identified in sperm from fathers of children with ASD in comparison to that of fathers of neurotypical children. These results collectively suggest a possible mechanism involving altered methylation of a significant number of neurologically relevant ASD risk genes for introducing epigenetic changes associated with environmental exposures into the sperm methylome. Such changes may provide the potential for transgenerational inheritance of ASD as well as other disorders.

Keywords: DNA methylation; Faroe Islands; autism; endocrine disrupting compounds; sperm.

FIGURE 1

Overview of workflow for this study. The blue blocks on the right outline the analytical workflow and programs (identified by yellow font) used to identify and characterize the DMRs.

FIGURE 2

Correlation between the concentrations of DDE with those of DDT (A) and the sum of the four most prevalent PCB congeners (B) in the serum of semen donors. The 4 specific PCB congeners are PCB 118, PCB 138, PCB 153, and PCB 183.

FIGURE 3

Relationship between serum concentrations of DDE and smoking status (A) or body mass index (BMI) (B). In B, the range of BMIs is shown for individuals determined to be in the first, second, and third tertiles with respect to levels of serum DDE. Samples from the first and third tertiles were used to represent low and high exposures, respectively, in the current methylation analyses. These data show essentially no difference in the range of DDE exposures between smokers and nonsmokers as well as no difference in the range of BMIs as a function of DDE exposure levels.

FIGURE 4

Relationship between serum concentrations of DDE and various sperm parameters, including sperm concentration and mobility. The motility terms “rapid”, “slow”, “very slow”, and “immobile” refer to the ability of sperm to move efficiently (which is required to reach an egg and achieve fertilization). The graphs show the % of sperm in each sample that exhibit the indicated motility as a function of DDE levels in serum.

FIGURE 5

Heatmaps (A,B) and Manhattan plots (C,D) depicting the results of the WGBS analyses. DMRs from the Discovery set (A) and Validation set (B) are shown in the heatmaps. All significant DMRs are represented above the horizontal lines in the Manhattan plots (C = Discovery set; D = Validation set).

FIGURE 6

SNORD115 block (*) on chromosome 15 showing significant genome-wide differential methylation between exposure groups after correction for multiple testing. The green bar identifies the location of the SNORD115 block located on chromosome 15, while the blue and red bars indicate the locations of the DMRs identified in this study and the prior study on sperm from fathers of children with ASD by Feinberg et al. (2015). The location of distinct SNORD115 repeats are also shown.

FIGURE 7

Significant gene ontology terms from GofuncR analyses of DMRs from discovery and validation cohorts. Specific p-values are from a meta p-value analysis of the least dispensable significant (p < 0.05) gene ontology terms.

FIGURE 8

Overlap of DDE DMR-associated genes among the discovery and validation sets and those in the SFARI Gene database of ASD risk genes.

FIGURE 9

Results of pyrosequencing analyses of DMRs associated with CSMD1 (A,B), RBFOX1 (C), and NRXN2 (D). The box plot (A) shows differential methylation at a single CpG site in CSMD1 while the graphs show the average methylation as a function of DDE serum concentration (μg/gm lipid) for CSMD1, RBFOX1 (10 sites, discovery set only), and NRXN2 (7 sites, all samples in both discovery and validation sets). R-squared (r²) and p-values for the correlation curves are shown.