Epigenome-wide association study (EWAS)

Multivariable linear regression was used to assess the differences in methylation at each measured CpG between (1) vapers versus non-smokers, (2) smokers versus non-smokers, and (3) smokers versus vapers, with adjustment for age, biological sex, BMI, educational attainment, household smoking, recreational drug use and 20 surrogate variables, using meffil [23]. We investigated CpGs which reached a Bonferroni-significance threshold of p < 5.91 × 10^–8 (0.05/846,244 CpGs tested), as well as a less stringent threshold of p < 1 × 10^–5. From these EWAS results, we identified differentially methylated regions (DMRs) using the dmrff R package [24]. DMRs were defined as regions containing at least two CpGs within 500 bp, each with EWAS meta-analysis p values < 0.05 and methylation changes in a consistent direction, and where the regional p value surpassed Bonferroni correction.

For the EWAS of vapers versus non-smokers, five additional models were run: (i) with adjustment for estimated cell type composition, (ii) with adjustment for self-reported smoking history (number of cigarettes), (iii) with adjustment for methylation at AHRR (cg05575921), an objective biomarker of smoke exposure [25], (iv) after excluding participants with < 60% salivary methylation at AHRR (cg05575921), indicative of a substantial smoking history [18], v) restricted to individuals of white ethnicity.

For the CpG sites identified in the EWAS of vapers versus non-smokers, and smokers versus non-smokers, we investigated whether there was evidence of a dose response in methylation levels based on the length of exposure history (6 months–1 year vs > 1 year for e-cigarette use, 6 months–5 years vs > 5 years for smoking).