Human exposure to trichloroethylene is associated with increased variability of blood DNA methylation that is enriched in genes and pathways related to autoimmune disease and cancer

Abstract

Human exposure to trichloroethylene (TCE) is linked to kidney cancer, autoimmune diseases, and probably non-Hodgkin lymphoma. Additionally, TCE exposed mice and cell cultures show altered DNA methylation. To evaluate associations between TCE exposure and DNA methylation in humans, we conducted an epigenome-wide association study (EWAS) in TCE exposed workers using the HumanMethylation450 BeadChip. Across individual CpG probes, genomic regions, and globally (i.e., the 450K methylome), we investigated differences in mean DNA methylation and differences in variability of DNA methylation between 73 control (< 0.005 ppm TCE), 30 lower exposed (< 10 ppm TCE), and 37 higher exposed ( $\ge$ 10 ppm TCE) subjects' white blood cells. We found that TCE exposure increased methylation variation globally (Kruskal-Wallis p-value = 3.75e-3) and in 25 CpG sites at a genome-wide significance level (Bonferroni p-value < 0.05). We identified a 609 basepair region in the TRIM68 gene promoter that exhibited hypomethylation with increased exposure to TCE (FWER = 1.20e-2). Also, genes that matched to differentially variable CpGs were enriched in the 'focal adhesion' biological pathway (p-value = 2.80e-2). All in all, human exposure to TCE was associated with epigenetic alterations in genes involved in cell-matrix adhesions and interferon subtype expression, which are important in the development of autoimmune diseases; and in genes related to cancer development. These results suggest that DNA methylation may play a role in the pathogenesis of TCE exposure-related diseases and that TCE exposure may contribute to epigenetic drift.

Keywords: DNA methylation; TCE; autoimmune disease; epigenetic variability; epigenetics; epigenome-wide association study (EWAS); occupational exposure.

Figure 1.

Global effect of human exposure to TCE. Box/scatter plots of the variance of methylation (M-value) values across all 399,439 CpG probes, categorized by varying levels of occupational exposure to TCE. Each point represents one subjects’ variance of global DNA methylation and the boxplots display the exposure-specific distribution of these points. Data was analyzed with the Kruskal-Wallis test and with Dunn’s post-hoc test. There were statistically significant differences in the variance of global DNA methylation between the TCE exposure groups (Kruskal-Wallis test p-value = 3.75e-3). The pairs that differed significantly from each other were the higher and lower TCE exposed (Dunn test Benjamini-Hochberg adjusted p-value = 0.0108) and the higher TCE exposed and controls (Dunn test Benjamini-Hochberg adjusted p-value = 0.0018).

Figure 2.

Differentially variable CpG probes (DVPs) in genes CTNND2 (a), EFNB2 (b), ZNF259 (c), and CAP2 (d) following varying levels of exposure to TCE and Manhattan plot of DVP results (e). The three boxplots displayed for each DVP (A-D) juxtapose the distribution of methylation values (β-values) with the levels of TCE exposure. A wide box is an indicator of increased variability of methylation, and a compressed box is indicative of decreased variability of methylation. The Manhattan plot (e) shows the negative log₁₀ p-value for each CpG probe when tested for differences in variability across the three comparison groups. The red horizontal line (–log₁₀(p) about 7) denotes the threshold for genome-wide significance (Bonferroni corrected p-value < 0.05) and the blue horizontal line (–log₁₀(p) around 4.5) denotes the threshold for DVPs that achieved a Benjamini-Hochberg corrected p-value < 0.05.

Figure 3.

Genome-wide significant differentially methylated region (DMR) comprised of 4 CpG probes and exhibiting hypomethylation with increased exposure to TCE (a). The DMR spans 610 basepairs on chromosome 11 and is located in the TRIM68 gene promoter (b). Also in this region is a CpG island and binding site for CTCF, an insulator protein that regulates chromatin opening and closing (b). The TRIM68 gene is represented by 13 CpG probes on the 450K array and, for each of these CpG probes, the TCE exposure-specific distribution of methylation values (β-values) is shown (a). The 4 probes emphasized in (a) are those that constitute the DMR (cg01505275, cg06925387, cg03122735, and cg16469099). The DMR was annotated with Ensembl version GRCh37.p13.

Figure 4.

Focal adhesion pathway with enriched genes DOCK1, FLNB, FLT1, FYN, IGF1R, PPP1R12A.. Analysis of biological pathways with WikiPathways considered a list of 219 candidate genes which matched to 288 significant DVPs that achieved Benjamini-Hochberg corrected p-value < 0.05 and did not match to East Asian superpopulation SNPs. Six of the candidate genes were found to be enriched in the focal adhesion pathway and are emphasized in the figure.