Steven A. Claas

BACKGROUND: Genetic research regarding blood lipids has largely focused on DNA sequence variation; few studies have explored epigenetic effects. Genome-wide surveys of DNA methylation may uncover epigenetic factors influencing lipid metabolism. METHODS AND RESULTS: To identify whether differential methylation of cytosine-(phosphate)-guanine dinucleotides (CpGs) correlated with lipid phenotypes, we isolated DNA from CD4+ T cells and quantified the proportion of sample methylation at >450 000 CpGs by using the Illumina Infinium HumanMethylation450 Beadchip in 991 participants of the Genetics of Lipid Lowering Drugs and Diet Network. We modeled the percentage of methylation at individual CpGs as a function of fasting very-low-density lipoprotein cholesterol and triglycerides (TGs) by using mixed linear regression adjusted for age, sex, study site, cell purity, and family structure. Four CpGs (cg00574958, cg17058475, cg01082498, and cg09737197) in intron 1 of carnitine palmitoyltransferase 1A (CPT1A) were strongly associated with very-low low-density lipoprotein cholesterol (P=1.8×10(-21) to 1.6×10(-8)) and TG (P=1.6×10(-26) to 1.5×10(-9)). Array findings were validated by bisulfite sequencing. We performed quantitative polymerase chain reaction experiments demonstrating that methylation of the top CpG (cg00574958) was correlated with CPT1A expression. The association of cg00574958 with TG and CPT1A expression were replicated in the Framingham Heart Study (P=4.1×10(-14) and 3.1×10(-13), respectively). DNA methylation at CPT1A cg00574958 explained 11.6% and 5.5% of the variation in TG in the discovery and replication cohorts, respectively. CONCLUSIONS: This genome-wide epigenomic study identified CPT1A methylation as strongly and robustly associated with fasting very-low low-density lipoprotein cholesterol and TG. Identifying novel epigenetic contributions to lipid traits may inform future efforts to identify new treatment targets and biomarkers of disease risk.

BACKGROUND: Although US cholesterol concentrations have dropped, &50% of adults have total cholesterol concentrations > or =5.18 mmol/L, putting them at "borderline-high risk" for heart disease. Whether the decline has continued into the 21st century is unknown. We assessed 20-year trends in cholesterol, hypercholesterolemia, lipid-lowering drug use, and cholesterol awareness, treatment, and control from Minnesota Heart Survey (MHS) data. METHODS AND RESULTS: Five independent, cross-sectional, population-based surveys of 2500 to 5000 adults were conducted in the Minneapolis-St. Paul, Minn, area from 1980 to 2002. Mean (nonfasting) total cholesterol concentrations have continued a 20-year decline, punctuated by an intervening lull. Age-adjusted mean total cholesterol concentrations in 2000 to 2002 were 5.16 and 5.09 mmol/L for men and women, respectively (in 1980 to 1982, 5.49 and 5.38 mmol/L for men and women, respectively) However, the decline has not been uniform across all age groups. Middle-aged to older people have shown substantial decreases, but younger people have shown little overall change and recently had increased total cholesterol values. The mean prevalence of hypercholesterolemia in 2000 to 2002 was 54.9% for men and 46.5% for women and has decreased significantly for both during the study. Age-adjusted mean high-density lipoprotein cholesterol concentrations in 2000 to 2002 were 1.09 and 1.40 mmol/L for men and women, respectively, and were not different from the prior survey. Lipid-lowering drug use rose significantly for both sexes aged 35 to 74 years. Awareness, treatment, and control of hypercholesterolemia have increased; however, more than half of those at borderline-high risk remain unaware of their condition. CONCLUSIONS: Although hypercholesterolemia prevalence continued to fall, significant population segments still have cholesterol concentrations near or at the level of increased risk.

DNA methylation is an important molecular-level phenotype that links genotypes and complex disease traits. Previous studies have found local correlation between genetic variants and DNA methylation levels (cis-meQTLs). However, general mechanisms underlying cis-meQTLs are unclear. We conducted a cis-meQTL analysis of the Genetics of Lipid Lowering Drugs and Diet Network data (n = 593). We found that over 80% of genetic variants at CpG sites (meSNPs) are meQTL loci (P-value<10(-9)), and meSNPs account for over two thirds of the strongest meQTL signals (P-value<10(-200)). Beyond direct effects on the methylation of the meSNP site, the CpG-disrupting allele of meSNPs were associated with lowered methylation of CpG sites located within 45 bp. The effect of meSNPs extends to as far as 10 kb and can contribute to the observed meQTL signals in the surrounding region, likely through correlated methylation patterns and linkage disequilibrium. Therefore, meSNPs are behind a large portion of observed meQTL signals and play a crucial role in the biological process linking genetic variation to epigenetic changes.

Recently, epigenetic age acceleration—or older epigenetic age in comparison to chronological age—has been robustly associated with mortality and various morbidities. However, accelerated epigenetic aging has not been widely investigated in relation to inflammatory or metabolic markers, including postprandial lipids. We estimated measures of epigenetic age acceleration in 830 Caucasian participants from the Genetics Of Lipid Lowering Drugs and diet Network (GOLDN) considering two epigenetic age calculations based on differing sets of 5′-Cytosine-phosphate-guanine-3′ genomic site, derived from the Horvath and Hannum DNA methylation age calculators, respectively. GOLDN participants underwent a standardized high-fat meal challenge after fasting for at least 8 h followed by timed blood draws, the last being 6 h postmeal. We used adjusted linear mixed models to examine the association of the epigenetic age acceleration estimate with fasting and postprandial (0- and 6-h time points) low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglyceride (TG) levels as well as five fasting inflammatory markers plus adiponectin. Both DNA methylation age estimates were highly correlated with chronological age (r > 0.90). We found that the Horvath and Hannum measures of epigenetic age acceleration were moderately correlated (r = 0.50). The regression models revealed that the Horvath age acceleration measure exhibited marginal associations with increased postprandial HDL (p = 0.05), increased postprandial total cholesterol (p = 0.06), and decreased soluble interleukin 2 receptor subunit alpha (IL2sRα, p = 0.02). The Hannum measure of epigenetic age acceleration was inversely associated with fasting HDL (p = 0.02) and positively associated with postprandial TG (p = 0.02), interleukin-6 (IL6, p = 0.007), C-reactive protein (C-reactive protein, p = 0.0001), and tumor necrosis factor alpha (TNFα, p = 0.0001). Overall, the observed effect sizes were small and the association of the Hannum residual with inflammatory markers was attenuated by adjustment for estimated T cell type percentages. Our study demonstrates that epigenetic age acceleration in blood relates to inflammatory biomarkers and certain lipid classes in Caucasian individuals of the GOLDN study. Future studies should consider epigenetic age acceleration in other tissues and extend the analysis to other ethnic groups.