A common and unstable copy number variant is associated with differences in Glo1 expression and anxiety-like behavior

Abstract

Glyoxalase 1 (Glo1) has been implicated in anxiety-like behavior in mice and in multiple psychiatric diseases in humans. We used mouse Affymetrix exon arrays to detect copy number variants (CNV) among inbred mouse strains and thereby identified a approximately 475 kb tandem duplication on chromosome 17 that includes Glo1 (30,174,390-30,651,226 Mb; mouse genome build 36). We developed a PCR-based strategy and used it to detect this duplication in 23 of 71 inbred strains tested, and in various outbred and wild-caught mice. Presence of the duplication is associated with a cis-acting expression QTL for Glo1 (LOD>30) in BXD recombinant inbred strains. However, evidence for an eQTL for Glo1 was not obtained when we analyzed single SNPs or 3-SNP haplotypes in a panel of 27 inbred strains. We conclude that association analysis in the inbred strain panel failed to detect an eQTL because the duplication was present on multiple highly divergent haplotypes. Furthermore, we suggest that non-allelic homologous recombination has led to multiple reversions to the non-duplicated state among inbred strains. We show associations between multiple duplication-containing haplotypes, Glo1 expression and anxiety-like behavior in both inbred strain panels and outbred CD-1 mice. Our findings provide a molecular basis for differential expression of Glo1 and further implicate Glo1 in anxiety-like behavior. More broadly, these results identify problems with commonly employed tests for association in inbred strains when CNVs are present. Finally, these data provide an example of biologically significant phenotypic variability in model organisms that can be attributed to CNVs.

Figure 1. Schematic representation of the duplication.

The top shows the location on chromosome 17 from 29.9 to 30.9 Mb and the duplication as identified by the HMM algorithm using hybridization intensity signals from probes (black circles) between B6 and A/J mice. The HMM predicted maximum and minimum boundaries are shown as red and green vertical lines. The duplicated region encompasses part of Btbd9, all of Glo1 and Dnahc8, and part of Glp1r. The location of primers for qPCR are shown as P1, P2, etc. red or green indicates which primer pairs were outside or inside the duplication, respectively. Forward primer 11 and reverse primer 3 or 4 were successfully used to amplify a fragment across the boundary of the duplication. S1 indicates the location of primers used for sequencing. Ten bases at each critical junction are shown; CTGA (underlined) is observed on either end of the duplication.

Figure 2. cis-eQTL for Glo1 and the probe 1458719_at.

Plot shows mouse genome on x-axis and log ratio score (LRS) on y-axis. Horizontal lines indicate genome-wide suggestive and significance thresholds as determined by permutation. Glo1 (1424109_a_at) (panel A) and the probe 1458719_at (panel B) measured by MOE430v2 microarray using hippocampal tissue as described elsewhere (generated by www.genenetwork.org).

Figure 3. Haplotype blocks from 30–31 Mb across 71 inbred strains at 48 SNP markers.

The duplicated region is denoted by heavy black vertical lines; duplicated strains are denoted with two repeating regions labeled ‘proximal copy’ and ‘distal copy’, whereas non-duplicated strains are indicated with a single box that contains the text ‘non-duplicated’. The major and minor alleles are coded as yellow (major) and blue (minor), respectively. Missing data at a given SNP is indicated in white. Strain names are shaded to indicate haplotype identity. Green boxes indicate haplotypes that contain the duplication. Red boxes (129X1/SvJ, BALB/cJ, BTBR_t+_tf/J, PERA/EiJ) indicate strains that belong to duplication containing haplotypes but do not have the duplication; we suggest that these strains have undergone reversions to the non-duplicated state. Within the duplicated region it was possible to genotype 4 SNPs (30,174,423; 30,174,589; 30,650,629; 30,650,736) that have been duplicated and to uniquely determine their genotype at both locations, as described in the text, these SNPs occur twice in the duplicated strains. Marker position (Build 36.1) and strain ID are indicated.

Figure 4. Relationship between behavior, duplication, gene expression and haplotype block.

A) hypothesized relationship between the duplication, Glo1 expression and anxiety like behavior; B) Correlation between duplication status and anxiety-like behavior as measured by percent time in the open field and presence of the duplication. A total of 901 male and female mice from 38 inbred strains were tested (n = 5–42 per strain). Random scatter along the x-axis and within duplicated and non-duplicated groups has been added. C) Correlation between duplication status and normalized gene expression in the amygdala from 27 inbred strains. Random scatter has again been added along the x-axis. D) Correlation between normalized Glo1 expression in the amygdala from 27 inbred strains and anxiety-like behavior as measured by percent time in the open field. E) Relationship between haplotype blocks and anxiety-like behavior as measured by percent time in the open field. Behavioral data were obtained from multiple individuals from each of the indicated number of strains, error bars represent the standard error for the mean of strains, not individuals, which would have been much smaller, but statistically inappropriate because the unit of analysis is strains not individuals.

Figure 5. Behavior and gene expression in CD-1 mice with and without the duplication.

There was significantly less activity, as measured by total distance traveled in the first 5 minutes in mice with the duplication (panel A). There was also significantly greater anxiety-like behavior, as measured as decreased percent time spent in the center of the open field in mice that had the duplication (panel B). Finally, there was a highly significant increase in Glo1 expression in whole brain homogenates of mice with the duplication. Expression was represented as fold change versus B6 mice, which are known not to carry the duplication; values were transformed to z-scores.