Clonal expansion of leukocytes harboring mosaic chromosomal alterations accelerates epigenetic aging and reshapes local DNA methylation

Mosaic chromosomal alterations (mCAs), a type of age-related clonal hematopoiesis, arise from postzygotic chromosomal gains, losses, or copy-neutral loss of heterozygosity (CN-LOH) in hematopoietic cells. DNA methylation serves as a molecular measure of biological aging and can be quantified through methylation-based epigenetic clocks. The extent to which mCAs accelerate epigenetic aging or induce local methylation remodeling is poorly understood. We analyzed 482 cancer-free participants from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial aged 54-77 at sample collection. Selection was based on mCA status (mCA carriers=261), prior genotyping, adequate DNA age and sex. Illumina MethylationEPIC array data raw IDATs were processed using the ChAMP pipeline with BMIQ normalization and ComBat batch correction. Six established methylation clocks (Horvath2013 and 2018, Hannum, PhenoAge, GrimAge, and DunedinPACE) were implemented using the dnaMethyAge R package to compute residual-based age acceleration. Multivariable linear models adjusted for age, sex, ancestry, smoking, and BMI compared mCA carriers to mCA-free individuals, as well as autosomal, mLOY, mLOX, and multi-mCA subtypes. Immune cell composition was estimated with EpiDISH using the IDOL-optimized FlowSorted.BloodExtended.EPIC reference. To identify local methylation effects, ordinary least squares regression tested per-CpG beta-values for differential methylation within mCA-affected regions and functional enrichment was performed using Ingenuity Pathway Analysis (IPA). Across six epigenetic clocks, mCA carriers showed higher epigenetic age acceleration vs mCA-free (e.g., PhenoAge beta=3.46, 95% CI 1.670-5.22, p=1.32x10 -4 ), with the largest effect sizes observed for autosomal mCAs. Methylation-based deconvolution of whole blood revealed shifts in leukocyte composition, including higher memory B-cell and lower CD4memory-cell proportions in participants with multiple mCAs (B-cell: beta=0.024, p=2.0x10 -8 ; CD4: beta=-0.017, p=0.017). Analyses of methylation levels at each CpG site within mCA regions identified 2,553 significant probes (1,973 hypo- and 580 hyper-methylated) with significant clusters visible across several CN-LOH and Gain regions. IPA revealed subtype-specific pathway perturbations, including growth-factor/GPCR signaling in Loss events , NAD biosynthesis and circadian regulation in CN-LOH, and suppressed interferon and TLR signaling in Gain events, with TGFB1 and TNF emerging as key upstream regulators. These findings suggest mCAs are associated with accelerated epigenetic aging, altered immune-cell composition, and localized epigenetic remodeling, with subtype-specific pathway disruptions that may reflect distinct compensatory mechanisms permitting clonal expansion in hematopoietic cells.