Epigenome-Wide Association Study for All-Cause Mortality in a Cardiovascular Cohort Identifies Differential Methylation in Castor Zinc Finger 1 (CASZ1)

Abstract

Background DNA methylation is implicated in many chronic diseases and may contribute to mortality. Therefore, we conducted an epigenome-wide association study (EWAS) for all-cause mortality with whole-transcriptome data in a cardiovascular cohort (CATHGEN [Catheterization Genetics]). Methods and Results Cases were participants with mortality≥7 days postcatheterization whereas controls were alive with≥2 years of follow-up. The Illumina Human Methylation 450K and EPIC arrays (Illumina, San Diego, CA) were used for the discovery and validation sets, respectively. A linear model approach with empirical Bayes estimators adjusted for confounders was used to assess difference in methylation (Δβ). In the discovery set (55 cases, 49 controls), 25 629 (6.5%) probes were differently methylated (P<0.05). In the validation set (108 cases, 108 controls), 3 probes were differentially methylated with a false discovery rate-adjusted P<0.10: cg08215811 (SLC4A9; log₂ fold change=-0.14); cg17845532 (MATK; fold change=-0.26); and cg17944110 (castor zinc finger 1 [CASZ1]; FC=0.26; P<0.0001; false discovery rate-adjusted P=0.046-0.080). Meta-analysis identified 6 probes (false discovery rate-adjusted P<0.05): the 3 above, cg20428720 (intergenic), cg17647904 (NCOR2), and cg23198793 (CAPN3). Messenger RNA expression of 2 MATK isoforms was lower in cases (fold change=-0.24 [P=0.007] and fold change=-0.61 [P=0.009]). The CASZ1, NCOR2, and CAPN3 transcripts did not show differential expression (P>0.05); the SLC4A9 transcript did not pass quality control. The cg17944110 probe is located within a potential regulatory element; expression of predicted targets (using GeneHancer) of the regulatory element, UBIAD1 (P=0.01) and CLSTN1 (P=0.03), were lower in cases. Conclusions We identified 6 novel methylation sites associated with all-cause mortality. Methylation in CASZ1 may serve as a regulatory element associated with mortality in cardiovascular patients. Larger studies are necessary to confirm these observations.

Keywords: cardiac biomarkers; epigenetics; mortality; outcome; transcriptome.

Figure 1

The Manhattan plot shows the results for the EWAS conducted on the discovery set. Probes that reached significance using a nominal P value of 0.05 were carried forward as candidates to be tested in the discovery set. CpG probes that are marked with a red X were those that were found to be significant using an FDR‐adjusted P value of 0.1 in the validation set. The bottom 3 panels show the 3 CpG probes that were significant in the validation set in the genome using UCSC genome browser tracks. The 3 CpGs identified are located within CpG islands and DNase clusters (ie, DNase hypersensitivity sites). The probe in CASZ1 (cg17944110) was located in the promoter/enhancer GH01J010790 (red track) and is in a region that is highly conserved. CASZ1 indicates castor zinc finger 1; EWAS, epigenome‐wide association study; FDR, false discovery rate; UCSC, University of California, Santa Cruz.

Figure 2

Plausible biological model displaying the consequences of increased methylation in the enhancer/promoter region of CASZ1, with the resulting changes in the expression of the target genes, UBIAD1 and CLSTN1, of GH01J010790. With increased methylation in the regulatory region in CASZ1, DNA becomes inaccessible to regulatory proteins such as transcription factors (TF). Therefore, expression of GH01J010790's target genes are decreased. With decreased UBIAD1 expression, cardiovascular morbidity and mortality are possibly increased through exaggerated vascular calcification, pathologic cardiac remodelling, oxidative stress, and increased HMG‐CoA availability, ultimately leading to increased all‐cause mortality in the cases. CASZ1 indicates castor zinc finger 1; HMG‐CoA, β‐hydroxy β‐methylglutaryl coenzyme A.