Epigenome-wide DNA Methylation Association Study of CHIP Provides Insight into Perturbed Gene Regulation

Abstract

With age, hematopoietic stem cells can acquire somatic mutations in leukemogenic genes that confer a proliferative advantage in a phenomenon termed "clonal hematopoiesis of indeterminate potential" (CHIP). How these mutations confer a proliferative advantage and result in increased risk for numerous age-related diseases remains poorly understood. We conducted a multiracial meta-analysis of epigenome-wide association studies (EWAS) of CHIP and its subtypes in four cohorts (N=8196) to elucidate the molecular mechanisms underlying CHIP and illuminate how these changes influence cardiovascular disease risk. The EWAS findings were functionally validated using human hematopoietic stem cell (HSC) models of CHIP. A total of 9615 CpGs were associated with any CHIP, 5990 with DNMT3A CHIP, 5633 with TET2 CHIP, and 6078 with ASXL1 CHIP (P <1×10^-7). CpGs associated with CHIP subtypes overlapped moderately, and the genome-wide DNA methylation directions of effect were opposite for TET2 and DNMT3A CHIP, consistent with their opposing effects on global DNA methylation. There was high directional concordance between the CpGs shared from the meta-EWAS and human edited CHIP HSCs. Expression quantitative trait methylation analysis further identified transcriptomic changes associated with CHIP-associated CpGs. Causal inference analyses revealed 261 CHIP-associated CpGs associated with cardiovascular traits and all-cause mortality (FDR adjusted p-value <0.05). Taken together, our study sheds light on the epigenetic changes impacted by CHIP and their associations with age-related disease outcomes. The novel genes and pathways linked to the epigenetic features of CHIP may serve as therapeutic targets for preventing or treating CHIP-mediated diseases.

Figure 1. Overview of Study Design

Figure 2. Genome-wide Directions of Effect of any CHIP and CHIP subtypes

a-d. Volcano plots with the effect size ( ) and −log₁₀(P) from the multiracial meta EWAS of (a.) any CHIP, (b.) DNMT3A CHIP, (c.) TET2 CHIP, and the EWAS in FHS of (d.) ASXL1 CHIP. Genes annotated to the CpG sites are shown.

Figure 3. Functional Validation in CRISPR/Cas9-edited HSCs Modeling CHIP

Dot plots of methylation status from −1.0 (no methylation) to 1.0 (complete methylation) seen in engineered primary cell cultures compared to correlation of EWAS results ranging from −0.1 to 0.1. Significance was determined with a binomial test. A) DNMT3A-associated CpG sites (n = 855 CpG sites) compared to DNMT3A engineered human stem cells(n=4). B) TET2-associated CpG sites (n = 312) compared to TET2-engineered human stem cells (n=4). C) ASXL1-associated CpG Sites (n = 139) compared to ASXL1-engineered human stem cells (n = 3).