Epigenomics and metabolomics reveal the mechanism of the APOA2-saturated fat intake interaction affecting obesity

Abstract

Background: The putative functional variant -265T>C (rs5082) within the APOA2 promoter has shown consistent interactions with saturated fatty acid (SFA) intake to influence the risk of obesity.

Objective: The aim of this study was to implement an integrative approach to characterize the molecular basis of this interaction.

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

Results: In the BPRHS, we 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 the GOLDN Study and the FHS. Additionally, in the FHS, cg04436964 methylation was negatively correlated with APOA2 expression in the blood of participants consuming a high-SFA diet. Furthermore, when consuming a high-SFA diet, CC carriers had lower APOA2 expression than those with the TT genotype. Lastly, metabolomic analysis identified 4 pathways as overrepresented by metabolite differences between CC and TT genotypes with high-SFA intake, including tryptophan and branched-chain amino acid (BCAA) pathways. Interestingly, these pathways were linked to rs5082-specific cg04436964 methylation differences in high-SFA consumers.

Conclusions: The epigenetic status of the APOA2 regulatory region is associated with SFA intake and APOA2 -265T>C genotype, promoting an APOA2 expression difference between APOA2 genotypes on a high-SFA diet, and modulating BCAA and tryptophan metabolic pathways. These findings identify potential mechanisms by which this highly reproducible gene-diet interaction influences obesity risk, and contribute new insights to ongoing investigations of the relation between SFA and human health. This study was registered at clinicaltrials.gov as NCT03452787.

Figures

Figure 1

Analysis scheme. Experiments, described in the leftmost column, proceeded from top to bottom. Cohort source and sample sizes for the various genotypes at APOA2 −265T >C under conditions of low or high SFA intake are given. For all analyses involving the BPRHS cohort, data from the same participants were used, and from the same data collection period. APOA2, apolipoprotein A-II; BPRHS, Boston Puerto Rican Health Study; FHS, Framingham Heart Study; GOLDN, Genetics of Lipid Lowering Drugs and Diet Network.

Figure 2

Close-up of the 81-kb genomic region surrounding the APOA2 −265T >C site (rs5082, the orange dot) of an epigenome-wide association study with APOA2 −265T >C in the BPRHS, GRCh37/hg19 approximate coordinates: chr1:161,134,000–1,612,215,000. (A) Under low saturated fat intake or <22 g/d (low-SFA), B) under high saturated fat intake or ≥ 22 g/d (high-SFA). The vertical axis displays the –log10(P value) of association, whereas the horizontal axis displays the physical position of CpG sites and genes (genome build 37). Each blue dot depicts one CpG site. The dashed line indicates the threshold of epigenome-wide significance at P = 1.1 × 10−7. ADAMTS4, a disintegrin and metalloproteinase with thrombospondin motifs 4; APOA2, apolipoprotein A-II; BPRHS, Boston Puerto Rican Health Study; B4GALT3, Beta-1,4-Galactosyltransferase 3; FCER1G, Fc fragment of IgE receptor Ig; NDUFS2, NADH:ubiquinone oxidoreductase core subunit S2; NR1I3, nuclear receptor subfamily 1 group I member 3; PPOX, protoporphyrinogen oxidase; TOMM40L, translocase of outer mitochondrial membrane 40 like.

Figure 3

Forest plots of meta-analysis of methylation differences at 3 methylation sites over 3 populations (BPRHS, GOLDN, and FHS) between CC and TT genotypes according to low- and high-SFA intake. The summary of statistics (β, SE, 95% CI, P value) of the meta-analysis are listed on each panel: (A) cg04436964 at low-SFA intake; (B) cg04436964 at high-SFA intake; (C) cg24429674 at low-SFA intake; (D) cg24429674 at high-SFA intake; (E) cg24847046 at low-SFA intake; (F) cg24847046 at high-SFA intake. BPRHS, Boston Puerto Rican Health Study; FHS, Framingham Heart Study; GOLDN, Genetics of Lipid Lowering Drugs and Diet Network.

Figure 4

Correlation between APOA2 mRNA expression in whole blood and methylation at cg04436964 in the FHS at exam 8. (A) Under low saturated fat intake or <22 g/d (low-SFA), (B) under high saturated fat intake or ≥22 g/d (high-SFA). The y-axis displays a normalized and log2-transformed signal of APOA2 mRNA expression adjusted for age, gender, and heterogeneity of cell type, whereas the x-axis displays the methylation level of cg04436964. APOA2, apolipoprotein A-II; FHS, Framingham Heart Study.

Figure 5

Box plot of APOA2 mRNA expression according to APOA2 –265T >C (rs5082) genotypes in the FHS at exam 8. The cyan box indicates participants with the CC genotype and the magenta box is for participants with the TT genotype, not taking medication for hypertension, dyslipidemia, or diabetes. Inside the box, the empty circle indicates means and the horizontal lines are medians. The bottom and top edges of the box indicate the range of values between the first and third quartiles (the 25th and 75th percentiles), whereas whiskers indicate the data range outside the box. Beyond the whiskers are the outliers. APOA2, apolipoprotein A-II; FHS, Framingham Heart Study.

Figure 6

CC homozygotes display perturbations in the tryptophan metabolism pathway. The metabolites highlighted in green were significantly (P < 0.05) decreased in CC genotype (cyan box) when compared with TT genotype (magenta box) with high-SFA intake. Box plots of 4 metabolites that met 2 criteria—1) differed between CC and TT genotypes, and 2) was associated with cg04436964—were shown according to APOA2 genotypes and SFA intakes. Inside the box, the empty circle indicates means and the horizontal lines are medians. The bottom and top edges of the box indicate the range of values between the first and third quartiles (the 25th and 75th percentiles), whereas whiskers indicate the data range outside the box. Beyond the whiskers are the outliers.