Characteristics of C57BL/6ByJ x 129P3/J F₂ mice fed low- and high-energy diets (Experiment 1).

<p>Values are mean±SD (minimum-maximum).</p><p>Age refers the age of the mouse at the end of the high-fat diet period.</p>*<p>Male versus female, p<0.05.</p

QTL Analysis of Dietary Obesity in C57BL/6byj X 129P3/J F₂ Mice: Diet- and Sex-Dependent Effects

<div><p>Obesity is a heritable trait caused by complex interactions between genes and environment, including diet. Gene-by-diet interactions are difficult to study in humans because the human diet is hard to control. Here, we used mice to study dietary obesity genes, by four methods. First, we bred 213 F₂ mice from strains that are susceptible [C57BL/6ByJ (B6)] or resistant [129P3/J (129)] to dietary obesity. Percent body fat was assessed after mice ate low-energy diet and again after the same mice ate high-energy diet for 8 weeks. Linkage analyses identified QTLs associated with dietary obesity. Three methods were used to filter candidate genes within the QTL regions: (a) association mapping was conducted using >40 strains; (b) differential gene expression and (c) comparison of genomic DNA sequence, using two strains closely related to the progenitor strains from Experiment 1. The QTL effects depended on whether the mice were male or female or which diet they were recently fed. After feeding a low-energy diet, percent body fat was linked to chr 7 (LOD = 3.42). After feeding a high-energy diet, percent body fat was linked to chr 9 (<i>Obq5</i>; LOD = 3.88), chr 12 (<i>Obq34</i>; LOD = 3.88), and chr 17 (LOD = 4.56). The Chr 7 and 12 QTLs were sex dependent and all QTL were diet-dependent. The combination of filtering methods highlighted seven candidate genes within the QTL locus boundaries: <i>Crx</i>, <i>Dmpk</i>, <i>Ahr</i>, <i>Mrpl28</i>, <i>Glo1</i>, <i>Tubb5</i>, and <i>Mut</i>. However, these filtering methods have limitations so gene identification will require alternative strategies, such as the construction of congenics with very small donor regions.</p></div

QTLs for percent body fat in C57BL/6ByJ×129P3/J F₂ mice (Experiment 1).

<p>Chr = chromosome. cM = centimorgan based on the experimental map. Marker = nearest LOD score peak. “Plus” refers to the allele that increases the trait value. Sex = sex-dependent by the criterion described in the text. For percent variance, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068776#pone-0068776-t004" target="_blank"><b>Table 4</b></a>. For locus boundaries, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068776#pone-0068776-t005" target="_blank"><b>Table 5</b></a>.</p>a<p>Overdominance means that phenotype of heterozygotes differs from phenotypes of both homozygotes. *p<0.05. **p<0.01.</p

Comparison of QTLs from genome scans of B6 × 129 F₂ mice fed high-energy diets (Experiment 1) with published data.

<p>Chr = chromosome. C57BL/6J x 129S1/SvImJ refers to the results of a similar study that interbred these strains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068776#pone.0068776-Su1" target="_blank">[36]</a>. C57BL/6ByJ x 129P3/J refers to the results reported here. If the locus boundaries overlap, the QTLs are considered the same. Other crosses that have overlapping obesity QTLs are shown with the strain conferring the allele that increases the trait value first. NA = not applicable.</p

Multiple regression analysis of variance for percent body fat in C57BL/6ByJ × 129P3/J F₂ mice fed low- and high-energy diets (Experiment 1).

<p><a href="mailto:Chr@Mb%E2%80%8A=%E2%80%8Achromosome/megabase" target="_blank">Chr@Mb = chromosome/megabase</a>. % Var = percent variance accounted for. Interactions are denoted by a semicolon. Degrees of freedom (df) are predicated on main effects and interactions.</p

Percent body fat of F₂ mice by sex and genotype (Experiment 1).

<p>Percent body fat of F₂ mice by sex and genotype (Experiment 1).</p

Percent body fat of F₂ mice by genotype of interacting loci detected in the pairwise genome scan (Experiment 1): chr 2 and 16 (upper left), chr 5 and 9 (upper right), and chr 3 and 13 (females, lower left; males, lower right).

<p>The x-axis shows genotype: BB, homozygous for C57BL/6ByJ alleles; PP, homozygous 129P3/J strain alleles; BP, heterozygous. Values are mean ± SEM. Groups that do not share a common subscript (a-f) significantly differed by post hoc testing.</p

Experimental design.

<p>In Experiment 1, B6 x 129 F₂ mice were fed a low- and then a high-fat diet. Body composition was measured at the end of each diet period, and QTL analyses were conducted for each diet condition. To narrow down a list of candidate genes that could account for the QTL, genotype association mapping (Experiment 2) was conducted using inbred strains and 4 million imputed SNPs. Differential gene expression analysis of tissues using microarrays (Experiment 3) indicated which genes in the QTL boundaries were differentially expressed between the parental B6 and 129 strains in liver, muscle, and adipose tissue. DNA genomic sequences from the QTL regions were compared between the parental strains (Experiment 4) to identify variants that might affect gene function. Results of all four experiments were compared to highlight genes identified by multiple methods.</p

Interval maps of chr 7 (low-energy diet) and chr 9, 12, and 17 (high-energy diet), which each contained a QTL identified in the genome-wide scan linked to percent body fat in C57BL/6ByJ x 129P3/J F₂ mice (Experiment 1).

<p>LOD score peaks are marked with arrows, and the associated locus boundaries are indicated by black bars below the peaks. Genetic locations (cM) are based on the experimental map from this genetic cross. Analysis of one- and two-QTL models indicated there are two peaks on chr 9. Significance thresholds are denoted by horizontal lines (for details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068776#pone-0068776-g002" target="_blank"><b>Figure 2</b></a>).</p

Genome-wide scans for the percentage of body fat (% body fat) of C57BL/6ByJ x 129P3/J F₂ male and female mice fed low- and then high-energy diet (Experiment 1).

<p>Horizontal lines represent suggestive linkage thresholds determined by 1000 permutation tests [threshold = 3.08 when mice were fed the low-energy diet (dashed lines) and 3.77 when fed the high-energy diet (solid lines)]. (A and B) Sex-dependent QTLs are determined by the difference between the LOD scores when sex is used as an additive versus a combined additive and interactive covariate. Significant differences between the additive (dashed line) and additive plus interactive (solid line) models after the low-energy (A) and high-energy (B) diets are indicated by arrows. (C) Diet-dependent effects were determined by the difference in LOD scores between each diet period (arrows). The y-axis lists chromosome numbers. All analyses included age and litter size as covariates, and the diet-specific analysis in C was conducted using sex as an interactive covariate. Details about individual QTLs are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068776#pone-0068776-t002" target="_blank"><b>Table 2</b></a>.</p