Genome-wide association study of behavioral, physiological and gene expression traits in outbred CFW mice

Abstract

Although mice are the most widely used mammalian model organism, genetic studies have suffered from limited mapping resolution due to extensive linkage disequilibrium (LD) that is characteristic of crosses among inbred strains. Carworth Farms White (CFW) mice are a commercially available outbred mouse population that exhibit rapid LD decay in comparison to other available mouse populations. We performed a genome-wide association study (GWAS) of behavioral, physiological and gene expression phenotypes using 1,200 male CFW mice. We used genotyping by sequencing (GBS) to obtain genotypes at 92,734 SNPs. We also measured gene expression using RNA sequencing in three brain regions. Our study identified numerous behavioral, physiological and expression quantitative trait loci (QTLs). We integrated the behavioral QTL and eQTL results to implicate specific genes, including Azi2 in sensitivity to methamphetamine and Zmynd11 in anxiety-like behavior. The combination of CFW mice, GBS and RNA sequencing constitutes a powerful approach to GWAS in mice.

Figure 1. Components of study

Each of the 4 panels illustrates a component of the study: (A) Behavioral testing and measurement of physiological traits; (B) Genotyping-by-sequencing (GBS); (C) Measurement of gene expression in brain tissues using RNA-Seq; (D) QTL mapping for physiological and behavioral traits, and for gene expression.

Figure 2. Genetic characteristics of CFW mouse population

(A) Density of GBS SNPs on autosomal chromosomes; (B) Mean LD (r²) decay rates estimated using frequency-matched SNPs, with MAF > 20%, in a 34^th generation AIL derived from LG/J and SM/J strains^,, heterogeneous stock (HS) mice bred for > 50 generations, the Hybrid Mouse Diversity Panel (HMDP), a panel of 30 inbred lab strains^,, Diversity Outbred mice, and CFW mice; (C) Treemix analysis summarizing genetic relationship between CFW mice and inbred strains in Wellcome Trust sequencing panel.

Figure 3. QTLs for physiological and behavioral traits

(A) Minimum p-values for association across all tested behavioral and physiological phenotypes (see Supplementary Table 1 and 2 for details). (B) Genome-wide scan for testis weight and (C) Pre-pulse inhibition in response to +12 dB pre-pulse. (D) Association signal for testis weight near the QTL on chromosome on 13. (E) Association signal for pre-pulse inhibition near the QTL on chromosome 7. Dotted red lines indicate thresholds (p<0.1) estimated via permutation tests.

Figure 4. Overview of eQTL mapping

(A) Color of each pixel in the matrix depicts the lowest p-value among all eQTLs using a 10 Mb × 10 Mb window. (B) Overlap of genes with eQTLs in the three brain tissues detected using the traditional cis-eQTL mapping method (not ASE). The permutation-based p-value threshold for each eQTL is 0.05. (C) Genome-wide scan for total locomotor activity on day 3 of the methamphetamine sensitivity tests. (D) Association signal for total locomotor activity in the QTL region on chromosome 9. (E) Association signal for expression of Azi2 in the striatum, in the same region as panel D. Dotted red lines indicate thresholds (p < 0.1) estimated via permutation tests.