Insertion-deletions in a FADS2 intron 1 conserved regulatory locus control expression of fatty acid desaturases 1 and 2 and modulate response to simvastatin

Abstract

The fatty acid desaturase genes (FADS1 and FADS2) code for enzymes required for synthesis of omega-3 and omega-6 long-chain polyunsaturated fatty acids (LCPUFA) important in the central nervous system, inflammatory response, and cardiovascular health. SNPs in these genes are associated with numerous health outcomes, but it is unclear how genetic variation affects enzyme function. Here, lymphoblasts obtained from Japanese participants in the International HapMap Project were evaluated for association of expression microarray results with SNPs in the FADS gene cluster. Six SNPs in the first intron of the FADS2 gene were associated with FADS1 expression. A 10-SNP haplotype in FADS2 (rs2727270 to rs2851682) present in 24% of the population was associated with lower expression of FADS1. A highly conserved region coinciding with the most significant SNPs contained predicted binding sites for SREBP and PPARγ. Lymphoblasts homozygous for either the major or minor haplotype were treated with agonists for these transcription factors and expression of FADS1 and FADS2 determined. Simvastatin and the LXR agonist GW3965 both upregulated expression of FADS1 and FADS2; no response was found for PPARγ agonist rosiglitazone. The minor haplotype homozygotes had 20-40% higher induction of FADS1 and FADS2 after simvastatin or GW3965 treatment. A 22 bp polymorphic insertion-deletion (INDEL) was found 137 bp downstream from the putative sterol response element, as well as a 3 or 1 bp INDEL 81-83 bp downstream. All carriers of the minor haplotype had deletions while all carriers of the major haplotype had insertions. Individuals carrying the minor haplotype may be vulnerable to alterations in diet that reduce LCPUFA intake, and especially responsive to statin or marine oil therapy.

Figure 1. Expression quantitative trait locus in FADS2 intron 1 and associated haplotype blocks in the Japanese HapMap population

(A) A Manhattan plot of -log(p-value) vs. distance in Kb is shown for the association of individual SNPs with FADS1 expression, with gene annotations above. Two LD blocks for this population are shown, with darkness of shaded cells representing the degree of correlation (R²) between pairs of SNPs. * Marked SNPs were associated with the ratio of arachidonic to linoleic acid (a measure of apparent total desaturase activity) in other Asian populations [11, 39]. (B) Haplotypes for each LD block are shown, with major alleles for each SNP in blue, and minor alleles in red. Recombination between blocks is depicted by the density of lines connecting individual haplotypes, and the multiallelic D′ (0.89).

Figure 2. Haplotype block in European CEU HapMap population

FADS2 intron 1 is contained within a large single LD block in the European HapMap population. SNPs associated with apparent FADS1 activity are in close proximity and in LD with the region highly associated with FADS1 expression in the Japanese population. Abbreviations: F1 = SNPs associated with apparent FADS1 activity, inferred from fatty acid product/substrate ratios [–13]; IQ = SNPs associated with IQ in breastfed children [9, 40]; CAD = SNPs associated with coronary artery disease and c-reactive protein levels [7].

Figure 3. Conserved region overlapping with significant SNPs in FADS2 intron 1

Zoomed-in detail of FADS2 intron 1 in the Manhattan plot of -log(p-value) vs. base position depicts association of SNPs in the Japanese HapMap population with FADS1 expression, with conserved regions depicted below as percent identity with the human sequence. The red box outlines a region overlapping with the area of highest significance that is conserved from zebrafish to humans, and contains predicted binding sites for SREBP and PPARγ.

Figure 4. Basal FADS1 and FADS2 expression and drug response in lymphoblasts homozygous for major or minor haplotypes

(A) FADS1 expression normalized to the major haplotype cells grown under basal conditions (ordinary growth media = 1). Under basal conditions, the minor haplotype homozygotes had significantly lower basal FADS1 expression than the major haplotype homozygotes, and this pattern persisted with rosiglitazone treatment. In contrast, simvastatin or GW3965 treatment upregulated expression in both genotypes such that they were no longer significantly different. (B) The same data as in (A) expressed as the change in FADS1 expression relative to drug treatment, normalized to vehicle treatment (not shown) for each genotype. Minor haplotype homozygotes confirm the findings in (A), showing significantly greater fold increase in FADS1 in response to simvastatin and GW3965 and no difference from major haplotype homozygotes in response to rosiglitazone. (C) FADS2 expression normalized to basal condition for the major haplotype is equivalent for the two haplotypes under basal conditions and for rosiglitazone. Simvastatin and GW3965 differentially upregulate haplotype FADS2 expression so that the minor haplotype is greater than the major haplotype. (D) The change in FADS2 expression relative to each genotype’s vehicle control is significant for simvastatin and GW3965. *p<0.05

Figure 5. Hypothesized model for bidirectional regulation of FADS1 and FADS2 through a sterol response element in FADS2 intron 1

Genetic variants near a putative SRE in the first intron of FADS2 are associated with altered response to SREBP-1c agonists. Known environmental and physiological factors modulating SREBP-1c activity are shown.