Date Published: September 28, 2017
Publisher: Public Library of Science
Author(s): Elaheh Rahbar, Hannah C. Ainsworth, Timothy D. Howard, Gregory A. Hawkins, Ingo Ruczinski, Rasika Mathias, Michael C. Seeds, Susan Sergeant, James E. Hixson, David M. Herrington, Carl D. Langefeld, Floyd H. Chilton, Ludmila Prokunina-Olsson.
Genetic variants near and within the fatty acid desaturase (FADS) cluster are associated with polyunsaturated fatty acid (PUFA) biosynthesis, levels of several disease biomarkers and risk of human disease. However, determining the functional mechanisms by which these genetic variants impact PUFA levels remains a challenge. Utilizing an Illumina 450K array, we previously reported strong allele-specific methylation (ASM) associations (p = 2.69×10−29) between a single nucleotide polymorphism (SNP) rs174537 and DNA methylation of CpG sites located in the putative enhancer region between FADS1 and FADS2, in human liver tissue. However, this array only featured 20 CpG sites within this 12kb region. To better understand the methylation landscape within this region, we conducted bisulfite sequencing of the region between FADS1 and FADS2. Liver tissues from 50 male subjects (27 European Americans, 23 African Americans) were obtained from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study, and used to ascertain the genotype at rs174537 and methylation status across the region of interest. Associations between rs174537 genotype and methylation status of 136 CpG sites were determined. Age-adjusted linear regressions were used to assess ASM associations with rs174537 genotype. The majority of CpG sites (117 out of 136, 86%) exhibited high levels of methylation with the greatest variability observed at three key regulatory regions–the promoter regions for FADS1 and FADS2 and a putative enhancer site between the two genes. Eight CpG sites within the putative enhancer region displayed significant (FDR p <0.05) ASM associations with rs174537. These data support the concept that both genetic and epigenetic factors regulate PUFA biosynthesis, and raise fundamental questions as to how genetic variants such as rs174537 impact DNA methylation in distant regulatory regions, and ultimately the capacity of tissues to synthesize PUFAs.
Polyunsaturated fatty acids (PUFAs) are vital for normal growth and development, serving as key structural components of biological membranes and modulating critical signal transduction events. In particular, long-chain PUFAs (LC-PUFAs) are precursors of bioactive metabolites, which have been implicated in several human diseases including cardiovascular disease, cancer, and inflammation [1–4]. Historically, the metabolic conversion and endogenous synthesis of LC-PUFAs from dietary PUFAs has been considered to be slow and relatively uniform in humans. Studies over the past decade [5–27] have demonstrated that genetic and epigenetic variations near and throughout the fatty acid desaturase (FADS) gene cluster (Fig 1) account for large variations in circulating and cellular LC-PUFA levels. Several common polymorphisms within the FADS gene cluster, including the single nucleotide polymorphism (SNP) rs174537, have been shown to be highly associated with circulating and tissue PUFA levels and PUFA product-to-precursor ratios [9–11, 28, 29]. In light of these studies, the conventional paradigm of slow, inefficient and uniform LC-PUFA biosynthesis in humans has been challenged.
DNA methylation throughout the sequenced region was characterized, and included 136 CpG sites that spanned the three key regulatory regions: (i) the FADS1 promoter region, (ii) the putative enhancer region and (iii) the FADS2 promoter region (Fig 1). DNA methylation analyses were successfully performed on all 50 individuals (27 European American and 23 African American) that passed quality control metrics. Overall, we observed similar characteristics between the European and African American groups, with the exception of BMI and hypertension. African Americans in this cohort were more hypertensive despite having lower BMI. Characteristics of the study cohort are provided in Table 1.
Omega-6 and omega-3 LC-PUFAs such as arachidonic acid (ARA) and docosahexaenoic acid (DHA) have long been recognized to have vital structural roles in cellular membranes, brain development and function, and inflammation [39–41]. Given this strong relationship between LC-PUFAs and human physiology and recent evidence demonstrating the significant impact of genomic variants, including rs174537, on LC-PUFA levels and related phenotypes, we conducted a more in-depth investigation of the methylation status of 136 CpG sites between FADS1 and FADS2 genes. Our goals were to 1) characterize the DNA methylation landscape within this potentially important regulatory area; 2) identify new ASM associations with genotype at rs174537; and 3) better understand the molecular mechanism by which genetic and epigenetic variations with the FADS cluster may influence LC-PUFA biosynthesis in human tissues.
In conclusion, this in-depth characterization of the region between FADS1 and FADS2 genes revealed eight key CpG sites that were associated with genotype at rs174537. These data validate our previous observation of ASM between rs174537 and the methylation status of cg27386326 located in the putative enhancer region. Additionally, seven new key CpG sites were identified, based on FDR significance. Taken together, these data show a significant association between the genomic region tagged by rs174537 and DNA methylation in the putative enhancer region. Further investigation is needed to determine if altering the DNA methylation landscape at this region can influence FADS1 and FADS2 gene expression levels, ultimately impacting metabolic conversion capacities and the overall synthesis of LC-PUFAs in humans. In addition, it is unknown if alterations in the DNA methylation landscape will preferentially benefit individuals with certain genotypes (e.g. GG vs. TT). Needless to say, this study highlights the importance of genetic and epigenetic factors that may strongly impact the capacity of tissues such as the liver to synthesize LC-PUFAs.