Experiment
An average mouse in midlife weighs between 25-30g, with about a gram of tissue in the largest adipose depot (gonadal). C57BL/6ByJ mice have heavier gonadal depots on average than do 129P3/J mice. The goal of the current study was to map QTL for gonadal adipose depot weight and to identify the underlying genetic variants using these contrasting inbred strains.
Previously, several QTL for gonadal depot weight were mapped using an (C57BL/6ByJ x 129P3/J)F2 intercross population (J:83139) The focus of the current study was to fine map one of these previously identified QTL, Adip20, on Chromosome 9. Congenic strains were bred using C57BL/6ByJ (B6) and 129P3/J (129) mice with the goal of identifying male breeders with a donor region that overlapped the QTL location of Adip20 on Chr 9 that was discovered in the original F2 intercross.
Congenic mice that had either zero or one copy of the 129-derived donor region were bred by mating inbred B6 female mice with heterozygous congenic males. The approach reduced maternal effects (all mothers were the same genotype) and reduced imprinting effects (only fathers contributed the donor region). Each congenic mouse was potentially genetically unique (because the paternal donor region could shorten due to meiotic recombination). Therefore, each congenic mouse was genotyped to ensure that the donor region breakpoints were defined. While most mice had full-length donor regions, some had partial (shorter) regions.
Simple sequence-length polymorphism markers were evaluated by polyacrylamide gel electrophoresis after polymerase chain reaction amplification by locus-specific primers. Single nucleotide polymorphisms (SNPs) were assayed at three locations. Mice were euthanized and the body of each mouse was weighed. The gonadal adipose depot was removed and weighed to the nearest 0.1g.
Two microarray experiments to quantify gonadal depot adipose tissue gene expression between congenic mice with and without the donor region (Experiments 1 and 2) were conducted. In Experiment 1, tissue from male mice from congenic strain 4 was selected because the donor region captured the previously identified Adip20 peak (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 there were N=6 mice in each genotype group, for a total of 12 mice. RNA was isolated from each gonadal adipose depot and measured genome-wide exon-by-exon expression using the Affymetrix Mouse Gene 1.0 ST Array.
All congenic mice were treated as one mapping population and a general linear model analysis of the log-transformed gonadal adipose depot weight was conducted for each marker, using marker genotype and strain as fixed factors and body weight as a covariate, and using a type 1 (sequential) sum of squares. A significant statistical threshold was defined as an level of 0.05 after Bonferroni correction for the number of markers (N = 148;log10(alpha/N) = 3.47) and a suggestive threshold as an level of 0.63 (log10(alpha/N) = 2.37).
Using the sequential method, congenic strains with group sizes N > 12/genotype/strain were analyzed [Table S5]. The 12 congenic strains that met the sample-size criterion were put into one of three groups for comparison; the groupings and comparison order were suggested by the results of the MST analysis [Fig 3A]. Within the groups, pairs of strains were compared, in the predetermined order, starting from comparison of the host strain (all mice without the donor fragment) and the strain with the smallest donor region [Fig. 3B]. The sequential analysis results indicated the presence of four QTLs [Fig 3C.], which were ordered from proximal to distal, starting with QTL1. For most of the QTLs, the 129-derived allele increased the weight of the gonadal adipose depot, but QTL3 had the opposite direction of effect. (All mention of mouse genome coordinates refer to GRCm38.)
QTL1, Adip20a (adiposity 20a) mapped to Chr 9:42779420-44142555;
QTL2, Adip20b (adiposity 20b) mapped to Chr 9:48789608-53884418;
QTL3, Adip20c (adiposity 20c) mapped to Chr 9:54242579-54286770; and
QTL4, Adip20d (adiposity 20d) mapped to Chr9:58262383-124595110. The location of Adip20d was refined, drawing on marginally significant mapping results from the original F2 intercross, to marker rs3721068 with a 10 Mb flanking region on Chr 9: 108915010-118915010.
Integrating many methods to isolate candidate genes failed to converge on a single candidate gene. While no single gene was implicated consistently, several had more evidence that others, for instance, Dixdc1 and Sik2, were differentially expressed in gonadal adipose depots and contained several putative regulatory and missense variants.
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