Adiposity QTL <i>Adip20</i> decomposes into at least four loci when dissected using congenic strains

<div><p>An average mouse in midlife weighs between 25 and 30 g, with about a gram of tissue in the largest adipose depot (gonadal), and the weight of this depot differs between inbred strains. Specifically, C57BL/6ByJ mice have heavier gonadal depots on average than do 129P3/J mice. To understand the genetic contributions to this trait, we mapped several quantitative trait loci (QTLs) for gonadal depot weight in an F₂ intercross population. Our goal here was to fine-map one of these QTLs, <i>Adip20</i> (formerly <i>Adip5</i>), on mouse chromosome 9. To that end, we analyzed the weight of the gonadal adipose depot from newly created congenic strains. Results from the sequential comparison method indicated at least four rather than one QTL; two of the QTLs were less than 0.5 Mb apart, with opposing directions of allelic effect. Different types of evidence (missense and regulatory genetic variation, human adiposity/body mass index orthologues, and differential gene expression) implicated numerous candidate genes from the four QTL regions. These results highlight the value of mouse congenic strains and the value of this sequential method to dissect challenging genetic architecture.</p></div

QTL confirmation from the consomic strains.

<p>Confirmation criterion–one or more sexes and one or more reciprocal strains differed in the expected direction using a p-value threshold adjusted for multiple testing (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141494#pone.0141494.t003" target="_blank">Table 3</a></b>). <i>Bdln4</i> and <i>Bdln5</i> are combined because they are both on chromosome 9.</p

QTLs detection in consomic mice: Average values of body size and composition measures in inbred and consomics strains.

<p>Body weight (top), body length (middle), and adiposity (bottom) in inbred and consomic strains (means ± SEM). Left panels: Strains with 129 genetic background. Right panels: Strains with B6 genetic background. Asterisks (*) indicate a nominal difference between consomic strain and its inbred host (p < .0.05), # indicates significant after correction for multiple testing (p<0.0056). ^~p = 0.0545. ^&borderline significance. ^§mice are heavier (top panel) but have similar gonadal weight, thus are leaner after adjustment for body weight.</p

Gonadal adipose depot.

<p>Anatomical location of the gonadal adipose depot in a male mouse.</p

Detection of gonadal adipose depot weight QTLs on chromosome 9 by association analyses of a pooled congenic population.

<p>(<b>A</b>) Location of the markers in Mb on mouse chromosome 9 (mChr9); the y-axis is the—log p-values (black line) obtained in a general linear regression model analysis with body weight and strain as covariates (red line, significant threshold; gray line, suggestive). We defined the QTL confidence intervals by two units of—log10 p-value drop from the peak (blue line). For QTL1-QTL4 (Q1-Q4), the shaded peak areas correspond to QTL regions defined by the sequential method. (<b>B and C</b>) Average weight of the gonadal adipose depot by genotype (B6/B6, B6/129; <b>B</b>) and Cohen’s <i>D</i> effect size (<b>C</b>) of <i>rs3723670</i>, the most associated marker (i.e., the marker with the lowest p value). *p<0.0001, difference between genotypes.</p

Extraction of differentially expressed genes.

<p>Filtering on a volcano plot of the microarray data analysis of the Experiment 1 (top) and Experiment 2 (bottom). In Experiment 1, we selected the gonadal adipose tissue from male mice from congenic strain 4 with the donor region captured QTL1, QTL2, and QTL3 (42.6 to 58.3 Mb). Half of the mice were heterozygous for the donor region (129/B6; N = 7), and half were littermates homozygous for the donor region (B6/B6; N = 7). Experiment 2 replicated Experiment 1 except with N = 6 mice in each genotype group, for a total of 12 mice. The customized volcano plots depict the estimated log₂-fold change (x-axis) and statistical significance (−log₁₀ p-value; y-axis). Each dot represents one gene. The green dots show genes with absolute log₂-fold change > 0.58 (i.e., 1.5-fold change) and false discovery rate < 0.05; the red dots show genes with false discovery rate < 0.05; the yellow dots show genes with absolute log₂-fold change > 0.58; the black dots show genes that did not reach these statistical thresholds. We label genes that passed any filters by name. Twenty differentially expressed genes are reproducible between Experiment 1 and 2, labeled with blue text and square boxes. Red stars show two genes that passed the filters in one but not both experiments yet are noteworthy because their human orthologues are associated with adiposity or body mass index in genome-wide association studies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188972#pone.0188972.t002" target="_blank">Table 2</a>). We summarize the additional experimental details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188972#pone.0188972.s012" target="_blank">S12 Table</a>.</p

Comparison of sex-specific QTLs between F₂ crosses and consomic strains.

<p>Differences between consomic and inbred host strains are indicated by:</p><p>x, p≥0.05 (no significant differences)</p><p>√, p<0.05 (a nominal p-value)</p><p>√√. p = 0.0545 (an adjusted p-value; <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141494#pone.0141494.t003" target="_blank">Table 3</a></b>).</p><p>QTL type was determined in F₂ crosses.</p

Inbred and consomic mouse strains used in this study.

<p>* Identification numbers (ID) are shown for strains available from the Jackson Laboratory (JAX; <a href="http://jaxmice.jax.org/" target="_blank">http://jaxmice.jax.org</a>) and the Mutant Mouse Regional Resource Center (MMRRC; <a href="https://www.mmrrc.org/" target="_blank">https://www.mmrrc.org</a>).</p><p>^# ‘Mon’ within mouse strain name is a laboratory code for the Monell Chemical Senses Center issued by the Institute for Laboratory Animal Research (ILAR; <a href="http://dels.nas.edu/ilar_n/ilarhome/labcode.shtml" target="_blank">http://dels.nas.edu/ilar_n/ilarhome/labcode.shtml</a>).</p><p>Abbreviations: F = female; M = male; M = mean; SD = standard deviation; d = days; n/a = not applicable</p

QTLs originally detected in F₂ hybrids.

<p>* Adiposity is defined as the weight of the gonadal adipose depot relative to body size [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141494#pone.0141494.ref019" target="_blank">19</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141494#pone.0141494.ref021" target="_blank">21</a>]. However a study using percent body fat [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141494#pone.0141494.ref022" target="_blank">22</a>] is included here for reference because both these fatness measures are closely related and thus informative.</p><p><i># cM</i> positions as provided in the original report.</p><p>^<i>§</i> formerly referred to as <i>Adip5</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141494#pone.0141494.ref019" target="_blank">19</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141494#pone.0141494.ref020" target="_blank">20</a>].</p><p>^ observed when mice were fed a high-fat diet (mice ate a low-fat mouse chow diet in the other studies referenced in this table).</p><p>F₂%var = % trait variance in F₂ explained by the QTL; estimates are not available (NA) for all QTLs from all prior studies. Ref = reference.</p

Human genome-wide associations within mouse QTLs for adiposity and body mass index.

<p>Human genome-wide associations within mouse QTLs for adiposity and body mass index.</p