Coronary artery calcification (CAC) is a marker of atherosclerosis and is associated with increased risk of coronary heart disease (CHD) mortality, especially in individuals with type 2 diabetes (T2D). While numerous studies have identified genetic loci involved in CAC, CHD, and T2D, the shared genetic architecture between these highly associated traits is still being understood. We compared the effects of 207 genetic variants that were previously identified as associated with CHD and/or CAC in 2,971 individuals with T2D and 13,022 non-diabetic controls utilizing whole genome sequencing generated by the National Heart, Lung, and Blood Institute’s Trans-Omics for Precision Medicine (TOPMed) program. Participants were from four race/ethnic groups, including European American, African American, Hispanic/Latinx, and East Asian. CAC was first log transformed, then further transformed through inverse rank-based normalization of the residuals accounting for age and sex. Linear mixed models accounting for relatedness, implemented in GENESIS, were used to test for interaction between each variant and T2D status. Analyses were adjusted for age, sex, study and the first eleven principal components. The genetic main and interaction effects were assessed in a joint test using a two degree of freedom model to determine if a CHD variant was associated with CAC, then further evaluated to determine if these variants had a significantly different effect in T2D cases versus controls. Using a significance threshold of P<2x10-4, we identified eighteen CHD variants associated with CAC according to the joint test, of which six had a statistically significant different effect in T2D cases and controls (rs6494488 near RBPMS2, rs7212798 in BCAS3, rs1321309 near PI16, rs668948 near APOB, rs840616 near CALCRL, rs12897 near FNDC3B). The association of five of these variants was stronger in cases than in controls, while for rs7212798 it was stronger in controls. While rs668948 has not previously been implicated in previous CAC GWAS, it lies nearest to APOB, which is a known driver of plaque development and subsequent atherosclerosis. Similarly, rs840616 lies near CALCRL, which is a part of a group of calcitonin receptors involved in the maintenance of calcium homeostasis. Overall, 85 of the CHD variants were nominally significant (p<0.05) for CAC according to the joint test, including 55 variants with nominally significant T2D interactions. These results highlight T2D as an important moderator of the association of CHD loci with subclinical atherosclerosis. Through our analysis, we identified potential new loci involved in the development of CAC in individuals with T2D.

Epigenetic clocks have shown that patterns of DNA methylation from blood cells are strongly correlated with chronological age. Those with accelerated methylation age (methylation age that is greater than expected for chronological age) are at higher risk for several diseases of aging and death, but the biological processes underlying such advanced epigenetic age are incompletely understood. Clonal hematopoiesis of indeterminate potential (CHIP) results from somatic mutations in blood stem cells and may be found in ~20% of the elderly. CHIP most commonly arises due to mutations in the DNA methylation altering enzymes, TET2 and DNMT3A, and also associates with increased risk of death, cancer, and cardiovascular disease. Whether CHIP associates with accelerated methylation age is unknown. We used methylation and whole genome sequencing data from several cohorts in TOPMed together comprising thousands of persons to show that CHIP is strongly associated with increased epigenetic age acceleration. The most consistent association is observed for intrinsic aging (2.8 ± 0.36 years, p < 2.5 x 10-14), which measures epigenetic aging that is independent of changes in cell composition, while a more variable association was seen with extrinsic aging (2.5 ± 0.46 years, p < 4.6 x 10-7), which captures epigenetic aging that is driven by changes in cell composition. We also analyzed the gene-specific effects of CHIP mutations on epigenetic aging, dividing our CHIP carriers into DNMT3A, TET2, and all other CHIP mutations. We found that the increase in intrinsic age acceleration seen in CHIP was very consistent across different genes, while the increase in extrinsic aging was lower with DNMT3A mutations and higher in TET2 mutations. The epigenetic clock software we used can also predict cell type composition and leukocyte telomere length (LTL) from methylation. We observed an increased predicted proportion of CD8+/CD28-/CD45RA- T-cells and decreased predicted LTL. Future experiments should seek to determine whether there is a causal relationship between CHIP and epigenetic age acceleration and whether intrinsic or extrinsic age acceleration is predictive of health outcomes in those with CHIP.