Fathers' preconception smoking and offspring DNA methylation

Abstract

Background: Experimental studies suggest that exposures may impact respiratory health across generations via epigenetic changes transmitted specifically through male germ cells. Studies in humans are, however, limited. We aim to identify epigenetic marks in offspring associated with father's preconception smoking.

Methods: We conducted epigenome-wide association studies (EWAS) in the RHINESSA cohort (7-50 years) on father's any preconception smoking (n = 875 offspring) and father's pubertal onset smoking < 15 years (n = 304), using Infinium MethylationEPIC Beadchip arrays, adjusting for offspring age, own smoking and maternal smoking. EWAS of maternal and offspring personal smoking were performed for comparison. Father's smoking-associated dmCpGs were checked in subpopulations of offspring who reported no personal smoking and no maternal smoking exposure.

Results: Father's smoking commencing preconception was associated with methylation of blood DNA in offspring at two cytosine-phosphate-guanine sites (CpGs) (false discovery rate (FDR) < 0.05) in PRR5 and CENPP. Father's pubertal onset smoking was associated with 19 CpGs (FDR < 0.05) mapped to 14 genes (TLR9, DNTT, FAM53B, NCAPG2, PSTPIP2, MBIP, C2orf39, NTRK2, DNAJC14, CDO1, PRAP1, TPCN1, IRS1 and CSF1R). These differentially methylated sites were hypermethylated and associated with promoter regions capable of gene silencing. Some of these sites were associated with offspring outcomes in this cohort including ever-asthma (NTRK2), ever-wheezing (DNAJC14, TPCN1), weight (FAM53B, NTRK2) and BMI (FAM53B, NTRK2) (p < 0.05). Pathway analysis showed enrichment for gene ontology pathways including regulation of gene expression, inflammation and innate immune responses. Father's smoking-associated sites did not overlap with dmCpGs identified in EWAS of personal and maternal smoking (FDR < 0.05), and all sites remained significant (p < 0.05) in analyses of offspring with no personal smoking and no maternal smoking exposure.

Conclusion: Father's preconception smoking, particularly in puberty, is associated with offspring DNA methylation, providing evidence that epigenetic mechanisms may underlie epidemiological observations that pubertal paternal smoking increases risk of offspring asthma, low lung function and obesity.

Keywords: DNA methylation; Epigenetic; Epigenome-wide association study; Paternal effects; Preconception; RHINESSA; Tobacco smoke.

Fig. 1

Manhattan plot for genome-wide distribution of dmCpGs. A: for father’s any preconception smoking and B: father’s pubertal smoking starting before age 15. The red line shows genome-wide significance, the blue is the suggestive line. The y-axis represents − log10 of the p value for each dmCpG (indicated by dots) showing the strength of association. The x-axis shows the position across autosomal chromosomes. The top dmCpGs on each chromosome were annotated to the closest gene

Fig. 2

Box plots showing distribution of methylation levels (beta-values) by significant dmCpGs sites in the EWAS. A: father’s any preconception smoking and B: for father’s pubertal smoking starting before age 15. The comparison p value between never-smoking exposed and smoking exposed offspring is shown above the box plot for each dmCpG

Fig. 3

Circos plots showing genome-wide distribution across autosomal chromosomes of dmCpGs associated with A personal smoking (in offspring), B mother’s smoking, C father’s any preconception smoking and D father’s pubertal smoking starting before age 15. Each dot represents a CpG site; the radial line shows the − log10 p value for each EWAS. Zoomed dots show CpG sites significant in at least one of the EWAS; each zoomed dot colour shows a unique CpG site specific locus

Fig. 4

Venn diagram showing EWAS CpG top hits for personal (offspring) smoking, mother’s smoking (FDR < 0.005), father’s any preconception smoking (top 100 dmCpGs) and father’s pubertal smoking starting before age 15 (FDR < 0.05) in the RHINESSA cohort, which are shared with top hits from meta-analysis of A PACE mother smoking (blue oval) as reported by Joubert et al. 2016 and B Personal cigarette smoking signature as reported by Christiansen et al. 2021 (blue) and by Joehanes et al. 2016 (yellow)

Fig. 5

Traits associated with the CpG sites that in EWAS were identified to be differentially methylated according to A father’s any preconception smoking, B father’s pubertal smoking starting before age 15

Fig. 6

Methylation effects on gene expression regulation across different tissue types for the CpG sites differently methylated according to father’s pubertal smoking starting before age 15 years (FDR < 0.05). [Accessed on 20 June 2021]. Size of point represents − log10 p value, colour scale shows CpG site correlation with expression; red to green represents increasing expression. In A shape shows the tissue type; in B shape shows genomic feature location

Fig. 7

Interactome of dmCpGs associated with father’s pubertal smoking. A String network at confidence score 0.7 and 20 top interactors (pale red nodes show dmCpGs, light green nodes show top interactors). The interactions show experimental evidence from score 0.0 (weak) to 1.0 (strong) using the colour gradient yellow (0.06) to deep purple (1.0). B Functional enrichment from UniprotKb for the top 15 biological processes, 10 molecular functions and 5 cellular components