Abstract Body (Do not enter title and authors here): Introduction/Background: CHIP is a risk factor for cardiovascular (CV) disease, cancer, and all-cause mortality. Previous work has shown that CHIP, and particularly larger CHIP clones, are associated with adverse CV outcomes, yet the molecular pathways through which CHIP impacts CV risk are poorly defined.

Hypothesis/Research Question: We hypothesize that the integration of whole blood transcriptomics and methylomics will provide novel insights into the pathophysiology of CHIP.

Methods/Approach: Whole blood DNA methylation profiling, transcriptomics, and whole exome sequencing with CHIP calling for variant allele frequencies (VAF) of ≥2% (CHIP) and ≥10% (large CHIP) were performed for 507 ISCHEMIA and ISCHEMIA-CKD participants with moderate-severe ischemia. We identified transcriptomic and methylomic differences between participants with CHIP and large CHIP vs no CHIP using DESeq2 and limma, adjusted for age, sex, and race/ethnicity. Gene set enrichment analysis (GSEA) and probe set enrichment analysis (PSEA) were performed to identify pathway-level alterations in transcription and methylation, respectively.

Results/Data: Clinical characteristics of study participants are described in Fig 1A. Compared to no CHIP (n=391), transcriptomics identified 6 differentially expressed genes (DEGs) in CHIP (n=116) and 27 DEGs in large CHIP (n=35) (p-adj<0.05; abs(logFC)>0.25) (Fig 1B). Compared to no CHIP, methylation identified no differentially methylated probes in CHIP and 6 in large CHIP (padj<0.20, abs(logFC)>0.03). GSEA identified 137 pathways significantly different in both CHIP and large CHIP vs. no CHIP (padj<0.05), while PSEA identified 724 and 2356 pathways (padj<0.20), respectively. Given its stronger relationship with methylation and transcription, downstream analyses focused on large CHIP. Integrating these data, we found 58 pathways to be both hypomethylated and transcriptionally upregulated in large CHIP, including azurophil granule-related pathways implicated in neutrophil degranulation (Fig 1C). Further investigation into the gene-probe pairs driving the azurophil granule pathway enrichment in large CHIP revealed hypomethylation and increased transcription of genes implicated in neutrophil extracellular trap formation, including ELANE, PTRN3, AZU1, and CTSG (Fig 1D).

Conclusions: Integration of the methylome and transcriptome suggests large CHIP is linked to neutrophil-mediated immune pathways in patients with stable coronary artery disease.