DNA Methylation and High-Density Lipoprotein Functionality—Brief ReportHighlights
The REGICOR Study (Registre Gironi del Cor)
Objective—The function of high-density lipoproteins (HDLs) may better reflect their atheroprotective role, compared with HDL-cholesterol levels. The association between DNA methylation and HDL function has not yet been established.
Approach and Results—We designed an epigenome-wide association study including 645 individuals from the REGICOR study (Registre Gironi del Cor). We determined DNA methylation from peripheral blood cells using the HumanMethylation450 array. We analyzed HDL functionality by determining HDL cholesterol efflux capacity and HDL inflammatory index. We discovered 3 methylation sites located in HOXA3, PEX5, and PER3 related to cholesterol efflux capacity and 1 located in GABRR1 related to HDL inflammatory index. Using a candidate gene approach, we also found 2 methylation sites located in CMIP related to cholesterol efflux capacity.
Conclusions—We identified 6 potential loci associated with HDL functionality in HOXA3, PEX5, PER3, CMIP, and GABRR1. Additional studies are warranted to validate these findings in other populations.
Low levels of high-density lipoprotein (HDL)-cholesterol (HDL-C) are associated with an increased risk of coronary heart disease (CHD). However, the causal relationship between HDL-C concentration and CHD is questioned by clinical trials and Mendelian randomization studies1,2; therefore, HDL function may better reflect its protective role. Two of the most important HDL functions, cholesterol efflux capacity (CEC) and HDL inflammatory index (HII), have been associated with the incidence of cardiovascular events and acute coronary syndromes.3,4 Finally, DNA methylation, the heritable and reversible addition of a methyl group to a cytosine–phosphate–guanine (CpG), is associated with HDL-C levels,5 but there is no evidence about its role on HDL functional properties.
Our aim was to assess the association between DNA methylation, CEC, and HII using an epigenome-wide association study and also exploring candidate genes.
Materials and Methods
Materials and Methods are available in the online-only Data Supplement.
Clinical characteristics of the 645 participants are available in Table I in the online-only Data Supplement. The correlation coefficient between the 2 analyzed HDL functions was −0.736 (P=0.010).
Epigenome-Wide Association Study Approach
The Manhattan and q–q plots are shown in Figures I through IV in the online-only Data Supplement. We identified 2 CpGs, Cg01964852 (HOXA3[homeobox A3]) and Cg22812457 (PEX5 [peroxisomal biogenesis factor 5]), associated with CEC in a model adjusted for confounders, surrogate variables, and HDL-C levels. We also identified the association between Cg01964852 (PER3 [period circadian clock 3]) and CEC in the model unadjusted for HDL-C levels. These 3 CpGs explained 5.38% of the CEC variability (Table). Finally, we found an association between Cg25671376 (GABRR1 [γ-aminobutyric acid–type A receptor, rho1 subunit]) and HII, explaining 0.83% of CEC variability (Table).
We looked at the public results of the CARDIoGRAMplusC4D consortium and identified variants in PER3, HOXA3, and GABRR1, showing suggestive associations with CHD (P=0.0006, 0.0027, and 0.0089, respectively; Table II in the online-only Data Supplement).
Candidate Gene Approach
Using the same individuals and a predefined selection of 2004 CpGs located in genes previously related to HDL-C levels (Table III in the online-only Data Supplement),5–7 we identified Cg08876518 and Cg03212183 (both in CMIP[c-Maf–inducing protein]) associated with CEC, in the model unadjusted for HDL-C levels (Table). These CpGs explained 3.70% of the CEC variability. No significant associations with HII were found.
The characteristics of the participants across methylation levels of the 6 CpGs of interest, the correlation between methylation levels, HDL functionality, and classical cardiovascular risk factors are shown in the Tables IV through XI in the online-only Data Supplement. We also analyzed the 2-way interactions between the CpGs and the CpGs and obesity and diabetes mellitus on HDL functionality and did not found any significant interaction (Tables XII through XIII in the online-only Data Supplement).
We identified 6 CpGs located in 5 genes—HOXA3, PER3, PEX5, CMIP, and GABRR1—showing differential methylation associated with HDL functionality: CEC or HII. CEC is the ability of HDLs to remove cholesterol excess from cells and has been related to hematopoietic stem cell mobilization and extramedullary hematopoiesis suppression, leading to decreased production of monocytes and neutrophils and lower atherosclerosis burden.8 A small proportion of CEC variability was explained by the methylation of CpGs located in HOXA3, PER3, PEX5, and CMIP. HOXA3 expression is associated with high abdominal adiposity,9 PER3 expression is altered in vascular smooth muscle cells in atherosclerotic plaques,10 PEX5 is a key gene in the peroxisome formation that is responsible for several lipid metabolism processes,11 and a CMIP polymorphism is associated with HDL-C levels.6,7 Methylation in these genes was inversely associated with CEC, independently of HDL-C levels.
HII is an indicator of HDL function related to the inflammatory process in atherogenic plaques.4 Hypomethylation of GABRR1 is associated with lower HII levels (more anti-inflammatory HDLs), although the variability explained is small. To the best of our knowledge, there is no relation between GABRR1 and lipid metabolism or functionality.
Genetic variants in PER3, HOXA3, and GABRR1 show potential associations with CHD in the CARDIoGRAMplusC4D consortium, suggesting a causal role of these genes in CHD development that could be mediated by HDL functionality.
Our study has some limitations. First, the cross-sectional design did not allow us to infer causality. Second, we could not replicate the results in an independent cohort; to mitigate this limitation, we explored public genome-wide association data (CARDIoGRAMplusC4D) to identify genetic variants in these loci that could be associated with CHD. Our major strength was the use of standardized protocols, the powerful statistical method, and the adjustment for residual confounding factors.
To summarize, we identified 5 loci potentially associated with HDL functionality in HOXA3, PEX5, PER3, CMIP, and GABRR1. This is the first evidence of associations between DNA methylation and HDL function. Further research to validate these associations is warranted.
We thank Elaine M. Lilly, PhD, for revising the English text.
Sources of Funding
This work was supported by Agència de Gestió d’Ajuts Universitaris de Recerca (2014-SGR-240), the Spanish Ministry of Economy through the Instituto de Salud Carlos III-FEDER (Fondo Europeo de Desarrollo Regional; CB06/03, CB12/03, PI12-00232, PI11-01801, FIS93/0568, and FIS92/0009-05), the Red de Investigación Cardiovascular (RD12/0042), the CIBER (Centro de Investigación Biomédica en Red) de Fisiopatología de la Obesidad y Nutrición (CIBEROBN) and the CIBER de Epidemiología y Salud Pública (CIBERESP). S.S.-B. and M.F. were funded by contracts from Instituto de Salud Carlos III-FEDER (IFI14/00007 and CES12/025, respectively), and A.H. received a fellowship from the Spanish Ministry of Education (FPU12/01318).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.116.308831/-/DC1.
- Received August 8, 2016.
- Accepted December 11, 2016.
- © 2017 American Heart Association, Inc.
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This is the first study to provide evidences of associations between DNA methylation and high-density lipoprotein function.
Hypomethylation of HOXA3, PEX5, PER3, and CMIP was associated with high cholesterol efflux capacity.
Hypomethylation of GABRR1 was associated with high high-density lipoprotein anti-inflammatory capacity.