Epigenomic and Metabolomic Signatures of APOA2 Gene by Saturated Fat Interaction
Epigenomic and Metabolomic Signatures of APOA2 Gene by Saturated Fat Interaction
Obesity is driven by genetic and environmental factors. Among the latter, diet is a most
 important one. The investigators refer to these combinations of genetic and dietary factors
 as 'gene-diet interactions.' Higher consumption of saturated fats (found mostly in foods of
 animal origin) has been associated with higher weight in people who were homozygotes for the
 minor allele at a genetic variant known as APOA2 -265 T>C (rs5082). In the current study, the
 investigators will seek to gain an understanding of the biological mechanisms driving this
 interaction. The investigators will select participants in three cohorts according to this
 genetic factor and conduct a series of molecular analyses (epigenetics, transcriptomics, and
 metabolomics). The analyses will identify epigenetic marks that are associated with saturated
 fat intake exclusively in subjects who carry this genetic factor. Moreover, the investigators
 will examine the association between epigenetic status and genotype at APOA2 and mRNA
 expression of the gene, and concentrations of metabolites in the blood. This study will
 increase the understanding of how genetics and diet act together to promote weight gain, and
 may eventually have implications for dietary recommendations that make use of genetic
 information.
Background: Apolipoprotein A-II (APOA2) is a significant constituent of high-density
 lipoproteins (HDL) with an undefined biological role. A putative functional variant, -265 T>C
 (rs5082) within the APOA2 promoter, has been shown consistently to interact with saturated
 fat (SFA) intake to influence the risk of obesity.

 Objective: This study will implement an integrative approach to characterize the molecular
 basis of this interaction.

 Design: The investigators will conduct an epigenome-wide scan on 80 participants carrying
 either the rs5082 less common genotype (CC) or the most common genotype (TT) and consuming
 either a low (<22 g/d) or high (≥22 g/d) SFA diet, matched for age, sex, BMI, and diabetes
 status in the Boston Puerto Rican Health Study (BPRHS). The investigators then will validate
 the findings in selected participants in the Genetics of Lipid Lowering Drugs and Diet
 Network (GOLDN) (n=379) and the Framingham Heart Study (FHS) (n=243). Transcription and
 metabolomics analyses will be conducted to determine the relationship between epigenetic
 status, APOA2 mRNA expression, and blood metabolites.

 Data sources. The Boston Puerto Rican Health Study (BPRHS), the Genetics of Lipid Lowering
 Drugs and Diet Network (GOLDN), and the Framingham Heart Study (FHS) Study selection: In
 BPRHS, 40 participants with CC genotype at APOA2 -265T>C (rs5082) will be selected, with 20
 reporting a low SFA intake (<22 g/d) and 20 indicating a high SFA intake (≥22 g/d). By
 matching age, sex, SFA intake, type 2 diabetes status, and BMI for the 40 participants with
 CC genotype, the second set of 40 participants with TT genotype of APOA2 -265T>C will be
 selected from the same population. In GOLDN, 107 participants with CC genotype and 272 with
 TT genotype for APOA2 -265T>C, who were not taking medication for hypertension, dyslipidemia,
 or diabetes, will be selected to validate the findings from BPRHS. In FHS, the investigators
 will include 73 unrelated participants with CC genotype and 170 with TT genotype at APOA2
 -265T>C, who did not take medication for hypertension, dyslipidemia, or diabetes.
 Participants of each genotype will be further divided into two subgroups based on SFA intake:
 low, <22 grams/day, high, ≥22 grams/day.

 Data Extraction. Genome-wide DNA methylation of isolated DNA samples in BPRHS and GOLDN was
 quantified using Illumina Infinium® human methylation 450K arrays (Illumina, San Diego, CA,
 USA). The methylation signal at each methylation site will be estimated as a β score, the
 proportion of total methylation-specific signal, and the detection P-value as the probability
 that total intensity for a given probe falls within the background signal intensity after
 normalization and quantity control check. FHS methylome data from dbGaP,
 accession#:phg000492.v2, where methylation statuses of 2,741 participants were measured in
 samples taken from participants attending their exam 8 visit between 2005 and 2008, using
 Illumina Infinium® human methylation 450K array. Methylation signals will be processed and
 normalized as for BPRHS and GOLDN. The investigators will obtain FHS transcriptome data from
 dbGaP under accession phe00002.v6. Metabolic profiling of plasma samples from those 80
 participants of BPRHS for whom the methylome analysis will be performed by Metabolon, Inc.
 (Durham, NC, USA) Outcomes: Body mass index will be the only outcome. Data Synthesis. (1)
 Epigenome-wide analysis of APOA2 genotype, by high and low SFA intake, will be conducted in
 80 matched case/control participants of BPRHS, replicated in selected participants of GOLDN,
 and FHS using linear mixed models. (2) A meta-analysis of the results from the three
 populations will be conducted using the meta R package.The comparison of allelic effect
 (beta) of APOA2 -265T>C from the meta-analysis between low and high SFA intake will be
 conducted with SAS 9.4 using a t-test. (3) Associations between epigenetic variants and APOA2
 genotypes with APOA2 mRNA expression will be tested in FHS. (4) Metabolome analysis will be
 conducted in the same 80 matched case/control participants of BPRHS, and metabolites will be
 correlated with epigenetic variants at APOA2 locus. (5) Enriched metabolic pathways will be
 examined in relation to identified epigenetic variants at APOA2 region.

 Knowledge translation plan: The results will be disseminated through interactive
 presentations at local, national, and international scientific meetings and publication in
 high impact factor journals. Target audiences will include the public health and scientific
 communities with interest in nutrition, diabetes, obesity, and cardiovascular disease.
 Feedback will be incorporated and used to improve the public health message and key areas for
 future research will be defined. Applicant/Co-applicant Decision Makers will network among
 opinion leaders to increase awareness and participate directly as committee members in the
 development of future guidelines.

 Preliminary findings: In BPRHS, the investigators identified methylation site cg04436964 as
 exhibiting significant differences between CC and TT participants consuming a high SFA diet,
 but not among those consuming low SFA. Similar results were observed in GOLDN and FHS.
 Additionally, in FHS, cg04436964 methylation was negatively correlated with APOA2 expression
 in participants consuming a high SFA diet. Furthermore, when consuming a high SFA diet, CC
 carriers had lower APOA2 expression compared to those with the TT genotype, but expression
 levels were similar when consuming a low SFA diet. Lastly, the metabolomic analysis
 identified four pathways as overrepresented by metabolite differences between CC and TT
 genotypes with high SFA intake.

 Significance: The investigators will apply multi-omics approaches to investigate the
 mechanistic foundations of the APOA2 -265T>C genotype by SFA interaction linked to the
 disruptive condition of obesity. the investigators may uncover plausible dysregulation of
 several metabolic pathways. This study will illustrate the effectiveness of multiple omics
 approaches to well-established gene-diet interactions, and contribute new evidence to ongoing
 explorations of the impact of saturated fat on human health.
Inclusion Criteria:

 -  subjects with the apoA2 genotype TT or CC

 Exclusion Criteria:

 -  all other genotypes