Scalable screening of ternary-code DNA methylation dynamics associated with human traits

David C Goldberg; Cameron Cloud; Sol Moe Lee; Bret Barnes; Steven Gruber; Elliot Kim; Anita Pottekat; Maximillian S Westphal; Luana McAuliffe; Elisa Majounie; Manesh Kalayil Manian; Qingdi Zhu; Christine Tran; Mark Hansen; Jelena Stojakovic; Jared B Parker; Rahul M Kohli; Rishi Porecha; Nicole Renke; Wanding Zhou

doi:10.1016/j.xgen.2025.100929

Scalable screening of ternary-code DNA methylation dynamics associated with human traits

Cell Genom. 2025 Jul 2:100929. doi: 10.1016/j.xgen.2025.100929. Online ahead of print.

Authors

David C Goldberg¹, Cameron Cloud¹, Sol Moe Lee¹, Bret Barnes², Steven Gruber², Elliot Kim¹, Anita Pottekat², Maximillian S Westphal², Luana McAuliffe², Elisa Majounie², Manesh Kalayil Manian², Qingdi Zhu², Christine Tran², Mark Hansen², Jelena Stojakovic², Jared B Parker³, Rahul M Kohli³, Rishi Porecha², Nicole Renke², Wanding Zhou⁴

Affiliations

¹ Center for Computational and Genomic Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
² Illumina, Inc., San Diego, CA 92122, USA.
³ Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
⁴ Center for Computational and Genomic Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address: wanding.zhou@pennmedicine.upenn.edu.

PMID: 40614726
DOI: 10.1016/j.xgen.2025.100929

Abstract

Epigenome-wide association studies (EWASs) are transforming our understanding of the interplay between epigenetics and complex human traits. We introduce the methylation screening array (MSA) to enable scalable and quantitative screening of trait-associated DNA cytosine modifications in large human populations. The MSA integrates EWASs and cell-type-linked methylation signatures, covering diverse traits and diseases. Using the MSA to profile the ternary-code DNA methylations-dissecting 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and unmodified cytosine-revealed a previously unappreciated role of 5hmC in mediating human trait associations and epigenetic clocks. We demonstrated that 5hmCs complement 5mCs in defining epigenetic cell identities. In-depth analyses highlighted the cell-type context of EWAS and genome-wide association study (GWAS) hits. Targeting aging, we uncovered shared and tissue-specific 5hmC aging dynamics and tissue-specific rates of mitotic hyper- and hypomethylation. These findings chart a landscape of the complex interplay of the two forms of cytosine modifications in diverse human tissues and their roles in health and disease.

Keywords: 5-hydroxymethylation; DNA methylation; Infinium array; epigenetic clock; epigenetics; epigenome-wide association study.