Robust validation of methylation levels association at CPT1A locus with lipid plasma levels

Abstract

There is increasing enthusiasm regarding the use of biobanked whole blood DNA as a model to discover methylation marks associated with biological phenotypes and generate novel mechanistic hypotheses ( 1 -3 ). DNA methylation has a critical role in cell functions and is cell-type specifi c. Such cell specifi city makes DNA methylation particularly challenging for epidemiological epigenetic investigations because disease relevant cell types might not be accessible due to practical issues such as availability, ethics, and cost associated with more complex specimen collection.Recent work suggests that agnostic methylation-wide association scan (MWAS) in peripheral blood can refl ect phenotype-associated methylation marks in other tissues and cell types, with effects detected in established effector cells much stronger than effects detected in blood ( 4 ). These observations suggest that marks detected in blood are associated with functions in effector cells. The Illumina HumanMethylation450 (HM450K) array is a robust assay to measure DNA methylation across the genome ( 4 -7 ). For any high-throughput technologies, and in particular for a novel assay such as the HM450K, rigorous quality control procedures are warranted and robustness of fi ndings must be validated through independent replication to avoid reporting spurious associations.In the current issue of the Journal , Frazier-Wood et al. ( 8 ) reported the novel fi ndings of signifi cant negative correlations between methylation levels at two CpG sites in the CPT1A locus and plasma levels of VLDL and LDL. Methylation levels were assessed in CD4+ T-cells isolated from peripheral blood DNA using the HM450K array. Given that no independent study samples were available for replication, to circumvent this challenge, the authors adopted an internal validation method by splitting the whole sample into " discovery and replication subsamples " . This strategy provides arguments in favor of the discovered associations but does not provide evidence of robustness against spurious findings due to sampling or confounding biases or any other undetected biases present in the study sample. A robust and thorough validation strategy implies the use of independent study samples and variation in the study designs ( 9 , 10 ). The validation phase is of particular importance in MWAS, as this technique is particularly subjected to confounders ( 3 ). Thus, we undertook to test for associations the two CTP1A CpG sites found associated with lipid-related traits by Frazier-Wood et al. using two independent study samples with considerable variations in their respective study design and with the design of the Frazier-Wood study.The studies had differences in sampling scheme, DNA methylation specimen, and array preprocessing approaches. The notable differences in the design and sample characteristics between the three studies are shown in Table 1 . Most notable is the method for lipid measurement, nuclear magnetic resonance spectroscopy in Frazier-Wood et al. and spectrophotometry in our stud...

The Epigenomic Analysis of Human Obesity

“…In fact, many of these same CpGs are associated with lipid‐related traits from specific EWAS performed for these measures, presumably because of their relationship to the development of adiposity or downstream‐induced epigenetic changes from BMI‐associated altered blood lipid profiles. Four of the ten DMPs were significantly related to triglyceride levels in a recent study by Dekkers et al , as well as in earlier and contemporary lipid studies . Furthermore, a study by Kriebel et al found that four of these CpGs are related to glucose‐related phenotypes .…”

Epigenetics of Lipid Phenotypes

“…Methylation at the top CPT1A locus explained 12% of TG variation in the discovery cohort (Genetics of Lipid Lowering Drugs and Diet Network (GOLDN), CD4+ lymphocytes, n=991) and 6% in the replication cohort (Framingham Heart Study, whole blood, n=1261) [12]. Subsequent studies have replicated the observed association with lipids [7, 15, 16] and lipoprotein subfraction profiles [17], as well as linked CPT1A methylation with obesity traits [18, 19], metabolic syndrome [20], and hypertriglyceridemic waist [21] in diverse populations. Interestingly, CPT1A loci were also shown to be differentially methylated in Dutch Hunger Winter survivors exposed to famine in utero , associating with both birth weight and serum LDL cholesterol levels later in life [22] and providing a possible mechanism for the well-known adverse metabolic sequelae of prenatal malnutrition.…”

SASH1, a new potential link between smoking and atherosclerosis

“…All subjects had a documented history of VT, were free of chronic diseases, and were free of inherited thrombophilia including: anti-thrombin, protein C and protein S deficiencies and homozygosity for the Factor V Leiden and Factor II G20210A mutations. For the methylation project, 349 MARTHA patients were randomly selected for DNA methylation analysis 173,174,175,176 .…”