Gene Expression By Pesticide Exposures during Gestation in the Charge Study, a Case-Control Investigation of Autism Spectrum Disorder

Background:   Pesticide exposures during gestation have been associated with increased ASD risk or symptoms in three populations. To our knowledge, the influence of prenatal xenobiotic exposures on gene expression in early childhood has not been previously explored. 

Objectives: 1) To identify genes that are differentially expressed in children living in households where pesticide sprays or foggers were used throughout the gestational period as compared with those not exposed to such applications; 2) To examine differences between children with autism spectrum disorder (ASD) vs. typically developing (TD) controls in pesticide-associated gene expression. 

Methods: The study included a multi-ethnic sample of 218 mother-child pairs from the Childhood Autism Risks from Genes and Environment Study (CHARGE) Study at the UC Davis MIND Institute with both RNAseq data and pesticide information. Children were aged 2-5 years. ASD diagnoses (n=145) were confirmed on the ADI-R and ADOS using DSM-5 criteria; controls from the general population were confirmed as having typical development (TD, n=73) based on the Mullen Scales of Early Learning, Vineland Adaptive Behavior Scales and Social Communications Questionnaire. An extensive interview with the mother collected information on household product use, including pesticide sprays and foggers. Frequent use was defined as one or more applications in each of at least 6 months during pregnancy.

Blood samples were drawn into PAX tubes after assessments and stored at -80 degrees until extraction of RNA, which was again frozen at -80. Sequencing was performed on the Illumina HiSeq 2000 platform.  Reads were aligned to hg19 using Bowtie and alignments were converted to a counts table using HTSeq. To address differences in sequencing depth between samples, the counts table was downsampled using binomial downsampling.  The association of gene expression with gestational exposures to pesticides was analyzed using a negative binomial generalized linear model in the Bioconductor package edgeR to identify significant up- or down-regulation. Analyses were planned in all mother-child pairs combined, and in ASD and TD groups separately, however, low prevalence of frequent pesticide use in TD families prohibited separate analysis of this group. The Comparative Toxicogenomics Database (CTD) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were searched for pathway and disease annotations associated with each gene. 

Results:  In both the full sample and the subset of children with ASD, hundreds of genes were differentially expressed comparing children from households with frequent vs. no pesticide applications during gestation. After FDR (false discovery rate) adjustment, 27 remained significant in each of those analyses. Among ASD children, over 96% of the top 50 differentially expressed genes were up-regulated, with only 4% down-regulated, in association with pesticide exposures.  Pathways linked to these over-expressed genes include thyroid hormone synthesis, wnt signalling, O-glycan biosynthesis and immune function. 

Conclusions:  Among 2-5 year old children with ASD, differential gene expression is associated with prenatal pesticide exposures, suggesting possible long-term potent impact on several pathways already linked to ASD, and on some novel ones.