High-resolution genetic mapping in the diversity outbred mouse population identifies Apobec1 as a candidate gene for atherosclerosis

Abstract

Inbred mice exhibit strain-specific variation in susceptibility to atherosclerosis and dyslipidemia that renders them useful in dissecting the genetic architecture of these complex diseases. Traditional quantitative trait locus (QTL) mapping studies using inbred strains often identify large genomic regions, containing many genes, due to limited recombination and/or sample size. This hampers candidate gene identification and translation of these results into possible risk factors and therapeutic targets. An alternative approach is the use of multiparental outbred lines for genetic mapping, such as the Diversity Outbred (DO) mouse panel, which can be more informative than traditional two-parent crosses and can aid in the identification of causal genes and variants associated with QTL. We fed 292 female DO mice either a high-fat, cholesterol-containing (HFCA) diet, to induce atherosclerosis, or a low-fat, high-protein diet for 18 wk and measured plasma lipid levels before and after diet treatment. We measured markers of atherosclerosis in the mice fed the HFCA diet. The mice were genotyped on a medium-density single-nucleotide polymorphism array and founder haplotypes were reconstructed using a hidden Markov model. The reconstructed haplotypes were then used to perform linkage mapping of atherosclerotic lesion size as well as plasma total cholesterol, triglycerides, insulin, and glucose. Among our highly significant QTL we detected a ~100 kb QTL interval for atherosclerosis on Chromosome 6, as well as a 1.4 Mb QTL interval on Chromosome 9 for triglyceride levels at baseline and a coincident 22.2 Mb QTL interval on Chromosome 9 for total cholesterol after dietary treatment. One candidate gene within the Chromosome 6 peak region associated with atherosclerosis is Apobec1, the apolipoprotein B (ApoB) mRNA-editing enzyme, which plays a role in the regulation of ApoB, a critical component of low-density lipoprotein, by editing ApoB mRNA. This study demonstrates the value of the DO population to improve mapping resolution and to aid in the identification of potential therapeutic targets for cardiovascular disease. Using a DO mouse population fed an HFCA diet, we were able to identify an A/J-specific isoform of Apobec1 that contributes to atherosclerosis.

Keywords: MPP; Multiparent Advanced Generation Inter-Cross (MAGIC); atherosclerosis; lipoproteins; multiparental models; multiparental populations; quantitative trait loci.

Figure 1

Overall design of the QTL mapping study. Mice obtained from the Jackson Laboratory were fed a chow diet of variable composition before arriving at the University of North Carolina Mouse Facilities at 4 wk of age. Mice were transferred to a controlled synthetic diet from 4 to 6 wk of age (AIN-76A). At 6 wk of age, clinical markers of cardiovascular disease were measured, and baseline QTL mapping was performed. The mice were then transferred to one of two diet groups such that one sibling of each sib pair was randomly assigned to either the high-fat, cholic acid diet group designed to induce atherosclerosis or the high-protein diet group, expected to be nonatherogenic. QTL mapping was then performed in 24-wk-old mice after diet exposure. Quantification of atherosclerotic lesions was performed in the mice at 24 wk of age. QTL, quantitative trait locus.

Figure 2

QTL mapping of clinical markers of cardiovascular disease in 6-wk-old DO mice at baseline. Genome-wide QTL scans for loci affecting plasma levels of triglycerides (A), total cholesterol (B), and glucose (C) in the DO population at baseline. Chromosomes 1 through X are represented numerically on the x-axis, and the y-axis represents the LOD score. The relative width of the space allotted for each chromosome reflects the relative length of each chromosome. Mice were maintained on a synthetic diet for 2 wk and then phenotyped for plasma clinical chemistries at 6 wk of age. Colored lines show permutation-derived significance thresholds (N = 1000) at P = 0.05 (LOD = 7.57, shown in red), P = 0.10 (LOD = 7.17, shown in orange), and P = 0.63 (LOD = 5.79, shown in yellow). QTL, quantitative trait locus; DO, Diversity Outbred; LOD, log of the odds ratio.

Figure 3

High-resolution mapping of significant hits on Chromosome 9 for plasma triglyceride levels. The eight coefficients of the QTL model show the effect of each founder haplotype on the phenotype. The model coefficients for the mapping of baseline triglycerides are plotted for each founder allele at each marker along Chromosome 9 and shading identifies the 95% Bayesian estimated interval around the peak (A). There are 34 potential candidate genes within the Chromosome 9 locus associated with plasma triglycerides at baseline (B). QTL, quantitative trait locus.

Figure 4

Effect of diet on total cholesterol in the DO mice. Genome-wide QTL scan for loci affecting plasma levels of total cholesterol after 18 wk of dietary treatment (A). Chromosomes 1 through X are represented numerically on the x-axis, and the y-axis represents the LOD score. The relative width of the space allotted for each chromosome reflects the relative length of each chromosome. Plasma was taken from 24-wk-old mice after 18 wk of dietary treatment. Colored lines show permutation-derived significance thresholds (N = 1000) at P = 0.05 (LOD = 7.57, shown in red), P = 0.10 (LOD = 7.17, shown in orange), and P = 0.63 (LOD = 5.79, shown in yellow). The eight coefficients of the QTL model show the effect of each founder haplotype on the phenotype. Shading identifies the 95% Bayesian credible interval around the peak (B). DO, Diversity Outbred; QTL, quantitative trait locus; LOD, log of the odds ratio.

Figure 5

QTL mapping of atherosclerosis in the DO mice. Genome-wide QTL scan for loci affecting atherosclerotic lesion size in mice fed a high-fat, cholic acid diet (A). Chromosomes 1 through X are represented numerically on the x-axis, and the y-axis represents the LOD score. The relative width of the space allotted for each chromosome reflects the relative length of each chromosome. Hearts were harvested from 146 mice after 18 wk on a high-fat, cholic acid diet. Colored lines show permutation-derived significance thresholds (N = 1000) at P = 0.05 (LOD = 7.57, shown in red), P = 0.10 (LOD = 7.17, shown in orange), and P = 0.63 (LOD = 5.79, shown in yellow). The eight coefficients of the QTL model show the effect of each founder haplotype on the phenotype. A/J founder alleles are associated with larger lesion size in the DO mice (B). There are six candidate genes within the 100,000-kb QTL interval on Chromosome 6: Apobec1, Gdf3, Dppa3, Nanog, Slc2a3, and the predicted gene Gm26168 (C). Gene expression data were obtained from livers from female C57BL6/J, A/J, NOD/ShiLtJ, NZO/HiLtJ, WSB/EiJ, CAST/EiJ, PWK/PhJ, and 129S1/SvImJ mice (http://cgd.jax.org/gem/strainsurvey26). Of the six candidate genes in the QTL interval on Chromosome 6, four were assayed by microarray for hepatic gene expression: Apobec1 (D), Gdf3 (E), Dppa3 (F), and Nanog (G). Apobec1 is the only candidate in this region that matches our allele effects such that A/J mice specifically express higher levels of Apobec1. QTL, quantitative trait locus; DO, Diversity Outbred; LOD, log of the odds ratio.

Figure 6

A/J preferentially expresses the long isoform of Apobec1 in response to a high-fat, cholic acid diet. Apobec1 expression levels of the short (A) and long (B) isoforms from RNA from liver tissue from A/J and C57BL/6J founder strain mice. A/J mice on a high-fat, cholic acid diet exhibit increased expression of both the long and short transcripts of Apobec1 in a diet-dependent manner, *P > 0.05. Apobec1 levels for each sample were normalized relative to RPS20. Fold changes are reported as the relative expression in A/J vs. C57BL/6J samples. Data are presented as mean ± SD, and significance was determined using a Student’s t-test.

Figure 7

Cis-eQTL for hepatic Apobec1 short and long isoforms in the DO mice. Genome-wide QTL scan for eQTL regulating expression of the short (A) and long (B) Apobec1 isoforms in the DO mice. Chromosomes 1 through X are represented numerically on the x-axis, and the y-axis represents the LOD score. The relative width of the space allotted for each chromosome reflects the relative length of each chromosome. Colored lines show permutation-derived significance thresholds (N = 1000) at P = 0.05 (LOD = 7.57, shown in red), P = 0.10 (LOD = 7.17, shown in orange), and P = 0.63 (LOD = 5.79, shown in yellow). The eight coefficients of the QTL model show the effects on the phenotype contributed by each founder haplotype on Chromosome 6 for mapping of the short (C) and long (D) isoforms of Apobec1. Shading identifies the 95% Bayesian credible interval around the peak. eQTL, expression quantitative trait loci; DO, Diversity Outbred; LOD, log of the odds ratio.

Figure 8

Apobec1 and ApoB levels are dependent on the genotype of UNC11996440, the Chromosome 6 peak SNP associated with atherosclerosis. Genotyping was performed using the Mega Mouse Universal Genotyping Array (MegaMUGA). Apobec1 mRNA levels were measured by quantitative polymerase chain reaction from liver tissue from the DO mice. Expression levels of the Apobec1 long isoform differed significantly between genotype classes (P > 0.001) (A). ApoB protein levels were measured in a subset of the mice phenotyped for atherosclerosis (N= 80 mice) using a mouse Apolipoprotein B Sandwich-ELISA method. ApoB expression levels differed significantly between the genotype classes (P > 0.001) (B).