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

Gonadal adipose depot.

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

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

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

The overlap of candidate gene by nomination method.

<p>We extracted candidate genes based on evidence from missense genetic variation, human adiposity, or body mass index orthologues and differential gene expression.</p

Characteristics of congenic mice by strains.

<p>Characteristics of congenic mice by strains.</p

QTL effects.

<p>The least square means (dark red lines) and 95% lower and upper confidence limits (blue lines) for QTL genotypes were obtained from the general linear model with body weight as covariate in the congenic mouse data used for the sequential analyses. We selected one marker in the middle of each QTL to plot each QTL effect. The host (A) comprised littermates homozygous for B6/B6 from all strains; we selected appropriate congenic mice (H) as suggested by the results of the sequential analysis.</p

Dissection of QTLs with the congenic strains and the sequential method.

<p>(<b>A</b>) Minimum spanning tree (MST). (<b>B</b>) Mean and standard error of the mean (SEM) by strain for gonadal adipose depot weight in grams. Host = combined group of homozygous littermates without the donor fragment from all congenic strains; N = number of mice in each group. P-values are for least-significant-difference t-tests for each sequential comparison; red text indicates p-values that met the threshold (p<0.05). For the comparisons of two strains, the first strain was either greater (>), less than (<), or equal to (=) the second strain in gonadal adipose depot weight. “Model” lists QTLs within the donor fragment of each congenic strain suggested by the sequential analysis. (<b>C</b>) Congenic strains and genotypes for informative markers: A = B6/B6, shown as white bars; H = 129/B6, shown as dark gray bars; the recombinant ends correspond to both sides of the donor fragment for each congenic strain between H and A (or chromosome end), shown as light gray bars. QTLs are shown at the bottom as black boxes; arrows show the direction of effect (e.g., ↑ indicates that the QTL allele from the 129 strain increases the trait value). The data support the presence of four QTLs; we reported the detailed logic for this conclusion in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188972#pone.0188972.s013" target="_blank">S1 Text</a>.</p