An APOO Pseudogene on Chromosome 5q Is Associated With Low-Density Lipoprotein Cholesterol Levels

Abstract

Background: Elevated levels of low-density lipoprotein cholesterol (LDL-C) are a major risk factor for cardiovascular disease via its contribution to the development and progression of atherosclerotic lesions. Although the genetic basis of LDL-C has been studied extensively, currently known genetic variants account for only ≈20% of the variation in LDL-C levels.

Methods: Through an array-based association analysis in 1102 Amish subjects, we identified a variant strongly associated with LDL-C levels. Using a combination of genetic analyses, zebrafish models, and in vitro experiments, we sought to identify the causal gene driving this association.

Results: We identified a founder haplotype associated with a 15 mg/dL increase in LDL-C on chromosome 5. After recombination mapping, the associated region contained 8 candidate genes. Using a zebrafish model to evaluate the relevance of these genes to cholesterol metabolism, we found that expression of the transcribed pseudogene, APOOP1, increased LDL-C and vascular plaque formation.

Conclusions: Based on these data, we propose that APOOP1 regulates levels of LDL-C in humans, thus identifying a novel mechanism of lipid homeostasis.

Keywords: cholesterol, LDL; chromosome mapping; founder effect; genetics; pseudogenes.

Figure 1. Recombination mapping of the LDL-C associated locus

A. SNPs within a 3.4 Mb region show strong association to LDL-C that span an extended haplotype. Enlarged version of Panel A provided as Supplemental Figure 1B. B. Recombination mapping based on individuals with partial LDL-C associated haplotypes narrows this region to a 442 kb region that includes 14 associated SNPs. The low and high LDL-C associated haplotypes are shown as white and black bars, respectively. LDL-C levels residualized following adjustment for age, sex, and APOB R3527Q genotype are normalized to subjects homozygous for the low LDL-C associated haplotype. See text for statistical comparisons between haplotype groups. C. The core haplotype, presumed to carry the risk variant. SNPs with a minor allele frequency <0.01 in the 1000G EUR are denoted by *.

Figure 2. Potential regulatory role of LDL-C associated SNPs

A. The region containing TIMD4 and APOOP1 is shown with SNP rs143855219 (red star) and possible chromatin looping. B. Coordinates of the two EcoRI fragments that are linearly approximately 10 kb apart. C. PCR product demonstrates potential chromatin looping of regions containing rs143855219 and APOOP1 promoter (lane 4). Lanes 1, 2, and 3: no cross-linked DNA, no restriction enzyme, and no DNA ligase controls, respectively. D. The 3C ligation junction sequences were confirmed via sequencing. E. Allele-specific transcription activity of another LDL-C associated SNP, rs898956003, (green star in A). The region containing the T allele has 1.9-fold higher transcriptional activity compared to the C allele (p=0.007).

Figure 3. LDL and plaque quantification in zebrafish

Quantification of (A) LDL-C levels (n=100 larvae per experiment) or (B) Average number of vascular lipid plaques (n=20 larvae per experiment) in larvae treated with either control or high cholesterol diet. Embryos were injected either with a MO to target orthologs for knockdown or with mRNA for overexpression of human genes. Results were compared to non-targeting control MO or ldlr MO. Data represent the average of three experiments. (*, p<0.01; student’s t-test with Bonferroni correction)

Figure 4. Suppression of ApoO impacts LDL-C and vascular lipid accumulation

Quantification of (A) LDL-C levels (n=100 larvae per experiment) or (B) average number of vascular plaques in larvae (n=20 per experiment) injected with MO targeting apoOb and fed either control or high cholesterol diet. Data represent averages across three experiments (*, p<0.01; student’s t-test with Bonferroni correction). (C) Representative brightfield (upper) and fluorescence (lower) microscopy images of caudal vein in larvae injected with either control MO, mRNA encoding human APOOP1, or apoOb MO and fed with high cholesterol diet containing cholesteryl conjugated BODIPY, observed in vascular plaques (arrowheads).