medRxiv. 2026 May 21. pii: 2026.05.14.26353250. [Epub ahead of print]
Charles E Breeze,
Gabriel A Goodney,
Hantao Wang,
Aubrey K Hubbard,
Jungeun Lim,
Mitchell J Machiela,
Thanh T Hoang,
Marie Richards-Barber,
Christine Tran,
Matthew Tolentino,
Mark Hansen,
Rishi Porecha,
Nicole Renke,
Wanding Zhou,
Nora Franceschini,
Sonja Berndt,
Jonathan Hofmann,
Mikyeong Lee,
Stephanie J London,
Jason Y Y Wong.
Tobacco smoking induces DNA methylation (DNAm) changes in blood and other tissues, which may influence chronic health outcomes. However, the breadth of smoking-related DNAm changes remains unmapped, offering a space for employing novel technologies. To expand our understanding of smoking impacts on DNAm, we conducted an epigenome-wide association study (EWAS) comparing ever smokers to never smokers, using blood from a multiethnic U.S. study population (n=887). We employed the newly developed Illumina Methylation Screening Array (MSA) covering 269,094 unique sites, including 123,776 CpGs not assayed in previous EWAS. Trans-ethnic meta-analysis identified 152 differentially methylated positions (DMPs) associated with ever-smoking status (n=764); European-specific analysis yielded 129 DMPs (n=674), including 106 overlapping with trans-ethnic analysis. A separate, large-scale replication EWAS (n=2,190) confirmed 91 trans-ethnic and 77 European-specific DMPs. Among our findings, we identified 61 DMPs at CpGs novel to the MSA platform, including near both new and known smoking-associated genes. Most notably, we uncovered a dense cluster of 12 DMPs within a 1117 bp region of ECEL1P1 , forming the most long-lasting, persistent smoking-associated DMR ever detected, even among former smokers who quit decades prior. We also detected new signals at AHRR , a well-known locus for smoking-related DNAm changes. eFORGE analysis revealed that detected smoking-associated DNAm changes are predominantly located in hematopoietic stem and progenitor cell (HSPC) DNase I hotspots, aligning with gene set enrichment analyses that highlighted pathways related to hematopoietic stem cell differentiation. Our findings suggest that HSPCs serve as a reservoir for an epigenetic memory of smoking. Additionally, we observed short-term cell-specific smoking-associated DNAm changes in myeloid cells. Our results demonstrate the utility of the MSA in expanding our knowledge of both transient and persistent environmental exposure-associated DNAm changes.
Highlights: Applied the state-of-the-art Methylation Screening Array (MSA), 269,094 unique sites including 123,776 not studied previously, which were selected for likely functional relevance.Identified 61 novel smoking-associated differentially methylated positions (DMPs), annotated to novel genes as well as genes previously associated with smoking-related DNA methylation. Smoking-associated DMPs were enriched in regulatory elements of hematopoietic stem and progenitor cells (HSPCs, via eFORGE analyses and GSEA) and HSPC regulatory genes (e.g. RUNX1 ), implicating HSPCs as reservoirs of long-term epigenetic memory. 12 DMPs collectively form the most long-lasting, persistent smoking-associated differentially methylated region (DMR) detected so far, spanning a 1117 bp region at ECEL1P1 . Smoking drives two distinct classes of DNAm alterations: transient, myeloid-specific changes and persistent, cell-type-shared signatures originating in HSPCs, forming a dual-track model of smoking-induced epigenetic remodeling.