DNA methylation and substance-use risk: a prospective, genome-wide study spanning gestation to adolescence

C A M Cecil, E Walton, R G Smith, E Viding, E J McCrory, C L Relton, M Suderman, J-B Pingault, W McArdle, T R Gaunt, J Mill, E D Barker, C A M Cecil, E Walton, R G Smith, E Viding, E J McCrory, C L Relton, M Suderman, J-B Pingault, W McArdle, T R Gaunt, J Mill, E D Barker

Abstract

Epigenetic processes have been implicated in addiction; yet, it remains unclear whether these represent a risk factor and/or a consequence of substance use. Here, we believe we conducted the first genome-wide, longitudinal study to investigate whether DNA methylation patterns in early life prospectively associate with substance use in adolescence. The sample comprised of 244 youth (51% female) from the Avon Longitudinal Study of Parents and Children (ALSPAC), with repeated assessments of DNA methylation (Illumina 450k array; cord blood at birth, whole blood at age 7) and substance use (tobacco, alcohol and cannabis use; age 14-18). We found that, at birth, epigenetic variation across a tightly interconnected genetic network (n=65 loci; q<0.05) associated with greater levels of substance use during adolescence, as well as an earlier age of onset amongst users. Associations were specific to the neonatal period and not observed at age 7. Key annotated genes included PACSIN1, NEUROD4 and NTRK2, implicated in neurodevelopmental processes. Several of the identified loci were associated with known methylation quantitative trait loci, and consequently likely to be under significant genetic control. Collectively, these 65 loci were also found to partially mediate the effect of prenatal maternal tobacco smoking on adolescent substance use. Together, findings lend novel insights into epigenetic correlates of substance use, highlight birth as a potentially sensitive window of biological vulnerability and provide preliminary evidence of an indirect epigenetic pathway linking prenatal tobacco exposure and adolescent substance use.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Differentially methylated loci at birth associated with adolescent substance use. (a) Manhattan plot showing genome-wide associations between DNA methylation at birth and later substance use (age 14–18). The dotted line represents the false discovery rate (FDR)-correction threshold (i.e., loci above the line are considered significant). (b) Prospective association between the top differentially methylated locus at birth and later substance use. The X axis shows substance use factor scores, whereas the Y axis represents beta methylation values, adjusted for sex and cell-type proportions. (c) Gene network analysis using GeneMANIA. Black circles represent genes (n=60) associated with the 65 probes found to be related to adolescent substance use in the genome-wide analysis at birth. Gray circles represent additional genes predicted by GeneMANIA based on genetic and physical interactions, shared protein domains as well as protein co-expression data. The gene network analysis demonstrates that, rather than being isolated, these genes clustered into a complex interconnected network. (d) Significantly enriched biological processes (blue), molecular functions (purple) and cellular components (red), based on gene ontology (GO) analysis of 60 genes annotated to probes that predict substance use at birth (n=65; q<0.05). Circles represent GO terms that survive FDR correction and contain at least one gene. The X axis represents −log(10) P-values. The opacity of the circles indicates level of significance (darker=more significant). The size of the circles indicates the percentage of genes in our results for a given pathway compared with the total number of genes in the same pathway (i.e., larger size=larger %).
Figure 2
Figure 2
Indirect effect of prenatal smoking on adolescent substance use via neonatal DNA methylation. (a) Path analytic indirect effects model. Dotted arrowed lines indicate non-significant paths. Single arrowed lines indicate standardized path coefficients that survived bootstrap-corrected confidence intervals (i.e., significant path). Red arrows show significant indirect path. Population effect sizes are interpreted using the standardized estimates (Std. B) following Cohen's guidelines: an effect of 0.10 is small effect, an effect of 0.24 is a medium effect, and an effect of 0.37 is a large effect. (b) Graphical representation of the indirect effect, where prenatal smoking associates with higher cumulative DNA methylation risk at birth (top panel), which in turn associates with higher substance use in adolescence (bottom panel). DNAm, DNA methylation.

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