Impact of Endocrine Disruptors on small RNA Expression in Sperm: a Risk Factor for Autism Spectrum Disorder?
Open AccessThe rapidly rising prevalence of autism spectrum disorders (ASD) in the U.S., now reported to be as high as 1 in 40 individuals, suggests that environmental factors may be increasing risk for ASD. However, there is a critical gap in our knowledge regarding the nature of environmental factors and the biological mechanisms through which they may influence susceptibility to ASD. Recent studies suggest that endocrine disrupting compounds (EDCs) may be contributing to the rising prevalence of ASD. Moreover, germline exposures to EDCs in animal models have been reported to induce lasting phenotypic changes in offspring which are, in some cases, transmitted transgenerationally and potentially through epigenetic mechanisms. The overarching goal of this study is to test the hypothesis that small RNA (smRNA) expression in sperm is associated with measured levels of 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (p,p’-DDE) in the serum of sperm donors from the Faroe Islands, whose diet rich in pilot whale meat and blubber predisposes them to higher than average levels of long-lived environmental EDCs, which accumulate in fatty tissues. Specifically, we investigated differences in the expression of miRNAs and piRNAs in sperm from men with the highest (third tertile) and lowest (first tertile) exposures with respect to blood DDE levels, and performed bioinformatics analyses on the differentially expressed miRNAs to identify transcriptional target genes and pathways that may predispose to ASD. Total sperm RNA was submitted for RNAseq analysis which employed a library preparation kit tailored for small RNAs (smRNA). RNAseq data was analyzed using the MicroRNA Analysis application (app) within Illumina’s BaseSpace Sequence Hub and different analytical pipelines in Galaxy and HIVE which are both web-based platforms for data intensive next-generation sequencing research. Then TargetScan or Ingenuity Pathways Analysis (IPA) software were used to identify the potential target genes of the differentially expressed miRNAs. The gene targets were then analyzed for pathways and functions using the Core Analysis feature of IPA. These targets were also overlapped with the Simons Foundation Autism Research Initiative (SFARI) Gene database to discover the genes that have an association with ASD. IPA analyses of gene targets revealed significant over-representation of pathways and functions relevant to neurological functions and disorders, including ASD.We also compared and contrasted different aligners in Galaxy and HIVE to discover whether different analytical pipelines resulted in the same differentially expressed smRNAs; certain smRNAs were identified by multiple aligners. Moreover, unsupervised cluster analyses of differentially expressed miRNAs and piRNAs using Multi-experiment Viewer software (MeV) showed that the high- and low-DDE exposure groups could be distinguished on the basis of the expression levels of these smRNAs. Therefore, the results of this pilot study show that differentially expressed microRNAs in sperm are associated with high levels of exposure to DDE in comparison to low exposures. These miRNAs potentially target genes involved in pathways and functions implicated in ASD.
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