Commercially available outbred mice for genome-wide association studies

Abstract

Genome-wide association studies using commercially available outbred mice can detect genes involved in phenotypes of biomedical interest. Useful populations need high-frequency alleles to ensure high power to detect quantitative trait loci (QTLs), low linkage disequilibrium between markers to obtain accurate mapping resolution, and an absence of population structure to prevent false positive associations. We surveyed 66 colonies for inbreeding, genetic diversity, and linkage disequilibrium, and we demonstrate that some have haplotype blocks of less than 100 Kb, enabling gene-level mapping resolution. The same alleles contribute to variation in different colonies, so that when mapping progress stalls in one, another can be used in its stead. Colonies are genetically diverse: 45% of the total genetic variation is attributable to differences between colonies. However, quantitative differences in allele frequencies, rather than the existence of private alleles, are responsible for these population differences. The colonies derive from a limited pool of ancestral haplotypes resembling those found in inbred strains: over 95% of sequence variants segregating in outbred populations are found in inbred strains. Consequently it is possible to impute the sequence of any mouse from a dense SNP map combined with inbred strain sequence data, which opens up the possibility of cataloguing and testing all variants for association, a situation that has so far eluded studies in completely outbred populations. We demonstrate the colonies' potential by identifying a deletion in the promoter of H2-Ea as the molecular change that strongly contributes to setting the ratio of CD4+ and CD8+ lymphocytes.

Figure 1. Ancestry of commercially available outbred stocks.

Most outbreds have a common origin: they descend from a single Swiss colony of 200 mice from which 2 males and 7 females were imported to the Rockefeller Institute for Medical Research in New York . Outbred Swiss stocks currently available include NMRI, CFW, MF1, CD1, ICR, NIHS, ND4 and SW. Not all outbreds descend from Lausanne in Switzerland. Non-Swiss strains include CF-1, NSA, OF1, SABRA and TO. Details on the origin of colonies are provided in Text S1.

Figure 2. Linkage disequilibrium decay radius (black) and minor allele frequencies (red) in outbred mice.

The scale of the vertical axis is megabases for the decay radius and ten times the value of the mean allele frequency (so a value of 2 is 0.2).

Figure 3. Proportion of laboratory inbred strain haplotypes found in commercial outbred stocks.

The region above the horizontal black line gives results from an analysis based on 351 markers from four regions in 66 colonies. Below the black line are results from a genome-wide analysis of 6 stocks. The degree of grey scale represents the contribution from each of the Perlegen re-sequenced strains to the outbred colonies.

Figure 4. Colonies, stocks, and ancestry.

Top two panels: relationship between colonies (top) and stocks (middle panel) shown by agglomerative clustering of Fst distances. Bottom panel: ancestry inferred from the FRAPPE program at K = 9. The length of each colored corresponds to the ancestry coefficient of each mouse, plotted along the horizontal axis. Mice are labeled by stock name (along the bottom) and by commercial provider along the top. Mice of the same colony were grouped together (giving rise to blocks of common ancestry, as seen for example to the right of the CD1 cluster) but individual colony labels omitted.

Figure 5. Association mapping of three phenotypes in three colonies.

The vertical scale is the negative logarithm (base10) of the P-value for the association; the horizontal scale is the position in megabases on the chromosome. On the left of the figure are results for ancestral haplotype reconstruction analysis (HAPPY) for all three colonies; on the right single marker association is shown for one colony for each phenotype: HsdWin:NMRI-NL for alkaline phosphatase; HsdWin:CFW-NL for high density lipoprotein and Crl:CFW(SW)-US_P08 for the CD4⁺/CD8⁺ T-cell ratio. LD structure around the associated SNP is shown by a red to white scale for r² = 0 to 1. For high density lipoprotein, each marker is represented by two diamonds. The right hand diamond of each pair is colored to show the r² with rs3476237 (at 173.6 Mb and the left hand the r² with rs3709584 (at 173.1 Mb). Gene annotations are taken from the UCSC genome browser.

Figure 6. A deletion in the promoter of the alpha chain of the MHC class II Eαβ heterodimer contributes to variation in the ratio of CD4⁺ to CD8⁺ T-lymphocytes.

On the left is shown PCR analysis of eight outbred mice demonstrating the presence of the deletion in the colony: smaller bands on the gel indicate animals with a homozygous deletion. On the right is shown the results of complementation with a transgene; the proportion of CD4+ and CD8+ cells was measured by flow cytometry in CD3+ splenocytes and CD3hi single-positive thymocytes of Eanull inbred NOD mice carrying the Ea16 complementing transgene (filled circles) or transgene-negative littermates (open circles).