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Lipoprotein subfractions help discriminate cardiometabolic disease risk. Genetic loci validated as associating with lipoprotein measures do not account for a large proportion of the individual variation in lipoprotein measures. We hypothesized that DNA methylation levels across the genome contribute to interindividual variation in lipoprotein measures. Using data from participants of the Genetics of Lipid Lowering Drugs and Diet Network (n = 663 for discovery and n = 331 for replication stages, respectively), we conducted the first systematic screen of the genome to determine associations between methylation status at ∼470,000 cytosine-guanine dinucleotide (CpG) sites in CD4+ T cells and 14 lipoprotein subfraction measures. We modeled associations between methylation at each CpG site and each lipoprotein measure separately using linear mixed models, adjusted for age, sex, study site, cell purity, and family structure. We identified two CpGs, both in the carnitine palmitoyltransferase-1A (CPT1A) gene, which reached significant levels of association with VLDL and LDL subfraction parameters in both discovery and replication phases (P < 1.1 × 10−7 in the discovery phase, P < .004 in the replication phase, and P < 1.1 × 10−12 in the full sample). CPT1A is regulated by PPARα, a ligand for drugs used to reduce CVD. Our associations between methylation in CPT1A and lipoprotein measures highlight the epigenetic role of this gene in metabolic dysfunction. Lipoprotein subfractions help discriminate cardiometabolic disease risk. Genetic loci validated as associating with lipoprotein measures do not account for a large proportion of the individual variation in lipoprotein measures. We hypothesized that DNA methylation levels across the genome contribute to interindividual variation in lipoprotein measures. Using data from participants of the Genetics of Lipid Lowering Drugs and Diet Network (n = 663 for discovery and n = 331 for replication stages, respectively), we conducted the first systematic screen of the genome to determine associations between methylation status at ∼470,000 cytosine-guanine dinucleotide (CpG) sites in CD4+ T cells and 14 lipoprotein subfraction measures. We modeled associations between methylation at each CpG site and each lipoprotein measure separately using linear mixed models, adjusted for age, sex, study site, cell purity, and family structure. We identified two CpGs, both in the carnitine palmitoyltransferase-1A (CPT1A) gene, which reached significant levels of association with VLDL and LDL subfraction parameters in both discovery and replication phases (P < 1.1 × 10−7 in the discovery phase, P < .004 in the replication phase, and P < 1.1 × 10−12 in the full sample). CPT1A is regulated by PPARα, a ligand for drugs used to reduce CVD. Our associations between methylation in CPT1A and lipoprotein measures highlight the epigenetic role of this gene in metabolic dysfunction. Cholesterol concentrations from lipoprotein fractions are some of the most commonly used clinical biomarkers of cardiovascular and metabolic disease risk (1.Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection. Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). 2001. JAMA. 285: 2486–97. online http://www.ncbi.nlm.nih.gov/pubmed/11368702 (Accessed May 22, 2013).Google Scholar). However, LDL cholesterol and HDL cholesterol concentrations do not account for all lipid-based CVD risk (1.Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection. Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). 2001. JAMA. 285: 2486–97. online http://www.ncbi.nlm.nih.gov/pubmed/11368702 (Accessed May 22, 2013).Google Scholar). In addition, the causal role of HDL cholesterol with CVD is debated (2Voight B.F. Peloso G.M. Orho-Melander M. Frikke-Schmidt R. Barbalic M. Jensen M.K. Hindy G. Hólm H. Ding E.L. Johnson T. Plasma HDL cholesterol and risk of myocardial infarction: a mendelian randomisation study..Lancet.380. 2012: 572-580http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3419820 286: 1468-1474Crossref PubMed Scopus (110) Google Scholar, 4Gray R.S. Robbins D.C. Wang W. Yeh J.L. Fabsitz R.R. Cowan L.D. Welty T.K. Lee E.T. Krauss R.M. Howard B.V Relation of LDL size to the insulin resistance syndrome and coronary heart disease in American Indians. The Strong Heart Study.Arterioscler. Thromb. Vasc. Biol. 1997; 17 (online PM:9409247.): 2713-2720Crossref PubMed Google Scholar, 5Festa A. Williams K. Hanley A.J. Otvos J.D. Goff D.C. Wagenknecht L.E. Haffner S.M. Nuclear magnetic resonance lipoprotein abnormalities in prediabetic subjects in the Insulin Resistance Atherosclerosis Study.Circulation. 2005; 111: 3465-3472Crossref PubMed Scopus (198) Google Scholar, 6Frazier-Wood A.C. Glasser S. Garvey W.T. Kabagambe E.K. Borecki I.B. Tiwari H.K. Tsai M.Y. Hopkins P.N. Ordovas J.M. Arnett D.K. A clustering analysis of lipoprotein diameters in the metabolic syndrome..Lipids Health Dis.10. 2011: 237http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3260106 52: 453-462Crossref PubMed Scopus (488) Google Scholar, 8Goff Jr, D.C. D'Agostino Jr, R.B. Haffner S.M. Otvos J.D. Insulin resistance and adiposity influence lipoprotein size and subclass concentrations. Results from the Insulin Resistance Atherosclerosis Study.Metabolism. 2005; 54: 264-270Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 9Syvanne M. Ahola M. Lahdenpera S. Kahri J. Kuusi T. Virtanen K.S. Taskinen M.R. High density lipoprotein subfractions in non-insulin-dependent diabetes mellitus and coronary artery disease.J Lipid Res. 1995; 36 (online PM:7775869): 573-582Abstract Full Text PDF PubMed Google Scholar). Currently, lipoprotein subfractions are limited in their clinical utility due to a lack of evidence that they convey information on the likelihood of experiencing an adverse cardiovascular event over and above that of traditional anthropometric, lipid, and demographic risk factors. However, identifying the genetic factors associated with lipoprotein subfractions may provide a more thorough understanding of the pathways that regulate lipoprotein composition and metabolism and so shed light on the etiology of atherosclerosis and IR. Numerous genetic loci have been associated with lipoprotein subfractions at genome-wide levels of significance, but these explain only a small percentage of individual variation in lipoprotein subfractions (10Chasman D.I. Paré G. Mora S. Hopewell J.C. Peloso G. Clarke R. Cupples L.A. Hamsten A. Kathiresan S. Mälarstig A. et al.Forty-three loci associated with plasma lipoprotein size, concentration, and cholesterol content in genome-wide analysis..PLoS Genet.5. 2009: 730http://dx.plos.org/10.1371/journal.pgen.1000730Google Scholar, 11Frazier-Wood A.C. Manichaikul A. Aslibekyan S. Borecki I.B. Goff D.C. Hopkins P.N. Lai C-Q. Ordovas J.M. Post W.S. Rich S.S. et al.Genetic variants associated with VLDL, LDL and HDL particle size differ with race/ethnicity..Hum. Genet.132. 2013: 405-413http://www.ncbi.nlm.nih.gov/pubmed/23263444Google Scholar). As yet, there have been no studies examining associations between lipoprotein subfractions and methylation patterns across the genome. We aimed to conduct the first epigenome-wide methylation study of 14 lipoprotein subfraction measures, to examine whether methylation fractions at cytosine-guanine dinucleotide (CpG) sites across the genome are associated with lipoprotein subfractions. Further, we aimed to combine high-resolution data on both common genetic variants and the methylation status of CpG sites under the hypothesis that any observed DNA methylation associations may be in part mediated by genotype. The original study population consisted of 1,328 men and women from 148 families consisting of a mix of familial relationships including the following: parent-offspring (N = 614), sibling (N = 667), grandparent-grandchild (N = 89), avuncular (N = 617), half-sibling (N = 22), grand avuncular (N = 69), half-avuncular (N = 23), first cousin (N = 268), half-grand avuncular (N = 12), first cousin once removed (N = 81), half-first cousin (N = 11), half-first cousin once removed (N = 4), and second cousin (N = 1). All participants were of European descent recruited in Minneapolis, Minnesota, and Salt Lake City, Utah. The primary aim of the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study was to characterize the role of genetic and dietary factors on an individual's response to both a high-fat meal challenge and fenofibrate intervention. The details of GOLDN have been published elsewhere (12Kabagambe E.K. Ordovas J.M. Tsai M.Y. Borecki I.B. Hopkins P.N. Glasser S.P. Arnett D.K. Smoking, inflammatory patterns and postprandial hypertriglyceridemia.Atherosclerosis. 2009; 203 (online PM:18804210): 633-639Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Briefly, the study protocol consisted of an initial screening visit (visit 0), during which participants were asked to discontinue the use of lipid-lowering drugs and over-the-counter medication that could affect lipid levels. Approximately 4 to 8 weeks later, baseline blood chemistries were measured (visit 1). A day later (during visit 2), participants' fasting (8 h fast) blood samples were collected. The final sample consisted only of those willing to undergo the high-fat meal protocol (N = 1,036 individuals) and who had useable NMR and genotype data after exclusions (see below; N = 817). The protocol was approved by the institutional review boards at the University of Minnesota, the University of Utah, Tufts University/New England and the University of at was from all All plasma samples used for this analysis were after a h All samples were for lipid once all were for each in the of VLDL, LDL, and HDL as as concentrations of each subfraction and were determined by NMR M.Y. A. Otvos J.D. Ordovas J.M. J.M. Arnett D.K. of and nuclear magnetic resonance in the of after an (online PubMed Scopus Google Scholar). NMR the by lipoprotein in the of a at The NMR is to of particle and concentrations for each of lipoprotein The particle for each lipoprotein LDL, and is as the of the lipoprotein particle diameters by the based on the of the NMR and in The of diameters for and large particle each of VLDL, LDL, and HDL are in for lipoprotein parameters as NMR not the of the NMR subclass in NMR a more of VLDL, LDL, and HDL and information subfraction that with that by J.D. Krauss R.M. of a nuclear magnetic resonance for plasma lipoprotein concentrations and from a PubMed Scopus Google Scholar). were with the to the that of lipoprotein measured by Lipoprotein from et Otvos J.D. Lipoprotein particle analysis by nuclear magnetic resonance Full Text Full Text PDF PubMed Scopus Google Scholar). in a from et Otvos J.D. Lipoprotein particle analysis by nuclear magnetic resonance Full Text Full Text PDF PubMed Scopus Google Scholar). 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Manichaikul A. Aslibekyan S. Borecki I.B. Goff D.C. Hopkins P.N. Lai C-Q. Ordovas J.M. Post W.S. Rich S.S. et al.Genetic variants associated with VLDL, LDL and HDL particle size differ with race/ethnicity..Hum. Genet.132. 2013: 405-413http://www.ncbi.nlm.nih.gov/pubmed/23263444Google Scholar). with and with the use of methylation as the to for these to the data in the of for the methylation to be for the as and the associations between methylation and these In methylation was the as to the of were to = that the use of methylation as a in of P The with methylation as the and lipoprotein as a is and but the to We for but do not on that lipoprotein subfraction measures are A.C. Glasser S. Garvey W.T. Kabagambe E.K. Borecki I.B. Tiwari H.K. Tsai M.Y. Hopkins P.N. Ordovas J.M. Arnett D.K. A clustering analysis of lipoprotein diameters in the metabolic syndrome..Lipids Health Dis.10. 2011: 237http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3260106 17 (online PM:9409247.): 2713-2720Crossref PubMed Google Scholar, 5Festa A. Williams K. Hanley A.J. Otvos J.D. Goff D.C. Wagenknecht L.E. Haffner S.M. Nuclear magnetic resonance lipoprotein abnormalities in prediabetic subjects in the Insulin Resistance Atherosclerosis Study.Circulation. 2005; 111: 3465-3472Crossref PubMed Scopus (198) Google Scholar, 6Frazier-Wood A.C. Glasser S. Garvey W.T. Kabagambe E.K. Borecki I.B. Tiwari H.K. Tsai M.Y. Hopkins P.N. Ordovas J.M. Arnett D.K. A clustering analysis of lipoprotein diameters in the metabolic syndrome..Lipids Health Dis.10. 2011: 237http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3260106 52: 453-462Crossref PubMed Scopus (488) Google Scholar, The LDL and the risk of coronary heart and (online Google Scholar, P. J. R. of and on LDL and composition. between and (online PubMed Scopus Google Scholar, A.C. Kabagambe E.K. Borecki I.B. Tiwari H.K. Ordovas J.M. Arnett D.K. evidence for an association between genotype and in 2012: Scholar). The role of VLDL in cardiometabolic disease been the of but this the role of CPT1A in an individual's lipoprotein subfraction CPT1A is in the and carnitine to so they be into for A. S. Y. W. in and response to status and in the 2013: Scholar, F.M. M. D. A. R. Y. A for in Lipid 2013: Scholar, H. Y. J. M. with metabolic and of in in type 2 2013: Scholar, but not in a in 2012: Scholar). of as which CVD CPT1A as do A. A. A. M. M. R. of that and the of gene CPT1A in J. 2011: Scholar, M. metabolic in of in lipid metabolism and response J. 2012: Scholar). Our that in methylation of CPT1A are associated with a lipid but these were not mediated by genetic variation in is not whether in methylation in across the genome. of CPT1A be associated with an in the of and a more lipid Genetic variation in CPT1A was not associated with lipoprotein diameters in analysis across two with any lipoprotein measures in a of genome-wide association study across (10Chasman D.I. Paré G. Mora S. Hopewell J.C. Peloso G. Clarke R. Cupples L.A. Hamsten A. Kathiresan S. Mälarstig A. et al.Forty-three loci associated with plasma lipoprotein size, concentration, and cholesterol content in genome-wide analysis..PLoS Genet.5. 2009: 730http://dx.plos.org/10.1371/journal.pgen.1000730Google Scholar, 11Frazier-Wood A.C. Manichaikul A. Aslibekyan S. Borecki I.B. Goff D.C. Hopkins P.N. Lai C-Q. Ordovas J.M. Post W.S. Rich S.S. et al.Genetic variants associated with VLDL, LDL and HDL particle size differ with race/ethnicity..Hum. Genet.132. 2013: 405-413http://www.ncbi.nlm.nih.gov/pubmed/23263444Google Scholar). in CPT1A been associated with lipid Hopkins S. Tiwari H.K. Genetic in carnitine gene are associated with variation in composition and fasting lipid in Lipid 2012: studies have to this K. A.C. M. S. D.I. et clinical and population of loci for blood PubMed Scopus Google and with M. S. D. J. fasting with a common carnitine 2011: Scholar). Thus, we that the association of genetic variation at this with cardiometabolic may be mediated by analysis not Our a association of methylation of CPT1A with VLDL and LDL parameters but be in the light of replication is a we methylation from CD4+ T which methylation A of studies on DNA methylation have that use of cells in studies in is and as a of but these studies have not lipoprotein S. G. of gene in blood cells is associated with insulin resistance in PubMed Scopus Google Scholar, S. A. a disease for DNA methylation PubMed Google Scholar, H. W.T. D. as epigenetic for mechanisms to of metabolic Full Text Full Text PDF PubMed Scopus Google Scholar). examine the of in the which may Our were to a population of European for A.C. Manichaikul A. Aslibekyan S. Borecki I.B. Goff D.C. Hopkins P.N. Lai C-Q. Ordovas J.M. Post W.S. Rich S.S. et al.Genetic variants associated with VLDL, LDL and HDL particle size differ with race/ethnicity..Hum. Genet.132. 2013: 405-413http://www.ncbi.nlm.nih.gov/pubmed/23263444Google Scholar). In addition, were and for each in of the in lipoprotein measure was not We studies that explain this we an association between methylation status at loci in CPT1A and the and the concentration of small LDL LDL and the and concentration of large and VLDL of these particles are associated with and that the of CPT1A is in insulin these may the methylation of CPT1A as a to reduce CVD risk. The are to the of the GOLDN study for the in data and with cytosine-guanine dinucleotide Genetics of Lipid Lowering Drugs and Diet Network insulin resistance methylation loci
Wood et al. (Tue,) studied this question.
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