Sensitized phenotypic screening identifies gene dosage sensitive region on chromosome 11 that predisposes to disease in mice

Abstract

The identification of susceptibility genes for human disease is a major goal of current biomedical research. Both sequence and structural variation have emerged as major genetic sources of phenotypic variability and growing evidence points to copy number variation as a particularly important source of susceptibility for disease. Here we propose and validate a strategy to identify genes in which changes in dosage alter susceptibility to disease-relevant phenotypes in the mouse. Our approach relies on sensitized phenotypic screening of megabase-sized chromosomal deletion and deficiency lines carrying altered copy numbers of ∼30 linked genes. This approach offers several advantages as a method to systematically identify genes involved in disease susceptibility. To examine the feasibility of such a screen, we performed sensitized phenotyping in five therapeutic areas (metabolic syndrome, immune dysfunction, atherosclerosis, cancer and behaviour) of a 0.8 Mb reciprocal chromosomal duplication and deficiency on chromosome 11 containing 27 genes. Gene dosage in the region significantly affected risk for high-fat diet-induced metabolic syndrome, antigen-induced immune hypersensitivity, ApoE-induced atherosclerosis, and home cage activity. Follow up studies on individual gene knockouts for two candidates in the region showed that copy number variation in Stat5 was responsible for the phenotypic variation in antigen-induced immune hypersensitivity and metabolic syndrome. These data demonstrate the power of sensitized phenotypic screening of segmental aneuploidy lines to identify disease susceptibility genes.

Figure 1. Sensitized phenotyping screen

Animals were subjected to a battery of tests designed to reveal effects of gene dosage on physiological parameters (grey boxes) relevant to common human diseases in five therapeutic areas: behaviour, metabolic syndrome, immune dysfunction, atherosclerosis, and cancer. Each therapeutic area was accompanied by a sensitizing environmental, pharmacological or genetic challenge (open boxes). The first cohort of mice were Apoe^KO/+ and were subjected to the full phenotyping battery, while a second cohort were Apc^Min/+ and were only tested for intestinal neoplasia-induced death. At 9 weeks of age, animals were tested for neophobia, gross activity and spatial learning in a home cage behavioural testing apparatus. At 12 weeks, blood parameters (cell counts and flow cytometry, cholesterol, glucose) were measured and at 14 weeks animals were tested for DNFB-induced CHS. At 15 weeks, animals were placed on a high-fat diet, and after 15 weeks of high-fat diet, blood parameters for clinical chemistry were collected and glucose tolerance measured. After 24 weeks of high fat diet, mice were sacrificed and organs collected. Aortic samples were examined for atherosclerotic plaques and bone marrow cells were extracted and analysed by flow cytometry (all dates are ±2 weeks).

Figure 2. Mouse lines carrying reciprocal Df and Dp on chromosome 11

1. Schematic representation of the segmental aneuploidy region on chromosome 11 containing 26 annotated and one unknown gene (mouse NCBI m37 assembly, April 2007, C57BL/6J reference strain) and syntenic region on human chromosome 17 (Human GRCh37 assembly February 2009). Df11(1)/+, WT and Dp11(1)/+ mice carried one, two and three copies, respectively, of genes in the interval.

2. CGH analysis of Df11(1)/+ versus WT and Dp11(1)/+ versus WT littermates confirmed expected gene dose changes across the rearrangement. Negative and positive log ratios indicate, respectively, loss and gain of genetic material.

Figure 3. Gene expression changes

Microarray hybridization analysis of expression patterns of genes within the rearrangement demonstrated changes in expression consistent with gene dosage. A heat map shows the normalized expression of genes in the Df11(1)/+ versus WT and Dp11(1)/+ versus WT samples. Average of two microarray experiments per sample (log₂ ratio for each gene was scaled with mean = 0 and SD = 1; log fold change and adjusted p values are indicated). Genes with a significant change in expression level are indicated (*p < 0.25).

Figure 4. Behavioural testing

1. A significant gene dose-dependent effect was observed for number of corner visits during the first 2 h after being placed in the monitoring apparatus (10 min intervals; mean ± SEM; Df11(1)/+, N = 22; WT, N = 45; Dp11(1)/+, N = 33).

2. No significant genotype effect on general day/night activity (number of corner visits) of animals was observed (3 h intervals; mean ± SEM; Df11(1)/+, N = 22; WT, N = 41; Dp11(1)/+, N = 24).

3. Significant gene dosage effect was observed on percentage of incorrect visits (2 h intervals). Dark and light phases are indicated by the dark and light bars, respectively (mean ± SEM; Df11(1)/+, N = 22; WT, N = 41; Dp11(1)/+, N = 24).

4. Significant gene dosage-dependent effect is observed on total numbers of nose pokes to incorrect corners (2 h intervals; mean ± SEM; Df11(1)/+, N = 22; WT, N = 41; Dp11(1)/+, N = 24; *Dp vs. Df, # Dp vs. WT and Df vs. WT; *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5. Peripheral blood cytometry

1. A,B. A significant positive gene dose-dependent effect was observed for (A) white blood cells, and (B) platelet counts at 12 weeks of age (mean ± SEM; Df11(1)/+, N = 21; WT, N = 41; Dp11(1)/+, N = 20; *P < 0.05, **P < 0.01, ***P < 0.001).

2. C–F. Flow cytometry revealed a significant positive gene dose-dependent effect for percentage (F) CD8+, but not (C) B220+, (D) Mac1+, (E) CD4+ positive cells in peripheral blood (mean ± SEM; Df11(1)/+, N = 19; WT, N = 58; Dp11(1)/+, N = 25; *P < 0.05, **P < 0.01).

Figure 6. Contact hypersensitivity and T cell function

1. A significant positive gene dose-dependent effect was observed for ear swelling following local application of DNFB. Ear thickness was measured at 24, 48 and 72 h following DNFB treatment in mice that had been sensitized by DNFB pre-treatment (mean ± SEM; Df11(1)/+, N = 22; WT, N = 42; Dp11(1)/+, N = 20; *p < 0.05, ***p < 0.001).

2. A significant positive gene dose-dependent effect was observed for the fraction of cultured naïve splenic T cells (Th0 cells) immunostaining for intracellular IL-17 (N = 4).

3. Quantitative real-time PCR revealed a positive gene dose-dependent effect on IL-17 mRNA expression in Th0 cells cultured for 5 days and stimulated acutely with cytokines. Data represents fold change compared to WT animals. N = 2 for each genotype.

4. The gene dose-dependent effect on fraction of IL-17+ T cells was occluded in cytokine-induced polarized T cells (Th17 cells, mean ± SEM, N = 4).

Figure 7. Metabolic parameters

1. A. Normal body weight in Df11(1)/+ and Dp11(1)/+ mice 4, 7, 10 and 13 weeks after initiation of high-fat diet (mean ± SEM; Df11(1)/+, N = 14; WT, N = 18; Dp11(1)/+ N = 11).

2. B. Significant negative gene dose-dependent effect on fasted blood glucose concentration in mice on normal (week 15), but not after 15 weeks of high-fat diet treatment (week 30).

3. C. Significant negative gene dose-dependent effect on glucose clearance following intra-peritoneal dextrose challenge after high-fat diet treatment for 15 weeks (AUC = area under the curve; Df11(1)/+, N = 20; WT, N = 36; Dp11(1)/+, N = 15 Controls were age-matched mice on a normal diet: Df11(1)/+, N = 7; WT, N = 19; Dp11(1)/+, N = 11 *p < 0.05).

4. D. Significant negative gene dose-dependent effect on plasma cholesterol in mice treated for 15 weeks with high-fat (week 30), but not normal diet (week 14 mean ± SEM; Df11(1)/+, N = 23; WT, N = 49; Dp11(1)/+, N = 22).

5. E,F. Significant positive gene dose-dependent effect on normalized (E) gonadal fat, but not (F) liver weight (mean ± SEM; Df11(1)/+, N = 20; WT, N = 35; Dp11(1)/+, N = 15). Weight of Gonadal fat and liver for each animal were normalized to total body weight.

Figure 8. Atherosclerotic aortic plaques

1. A. Representative histological sections of proximal aorta showing significant staining of plaque material (red, indicated by arrows) in all genotypes. Opposite gene dose-dependent effects on relative area of aortic plaques were seen in females (Df11(1)/+, N = 6; WT, N = 10; Dp11(1)/+, N = 3) and males (Df11(1)/+, N = 6; WT, N = 10; Dp11(1)/+, N = 5). In females, Df11(1)/+ mice showed significantly smaller plaques that WT mice, whereas in males, Df11(1)/+ mice showed a trend for increased plaque size compared to Dp11(1)/+ mice (mean ± SEM, *p < 0.05).

2. C. Kaplan–Meier survival plot of Apc^Min/+ animals with Df11(1) or Dp11(1) alleles and their WT littermates. No gene dose-dependent moderation of survival in Apc^Min/+ mice was observed. (Df11(1)/+; Apc^Min/+, N = 7; +/+; Apc^Min/+, N = 9; Dp11(1)/+; Apc^Min/+, N = 5).

Figure 9. Contact hypersensitivity in heterozygous Stat5ab and Stat3 knockout mice

1. A. A significant reduction of ear swelling was observed in heterozygous Stat5ab^KO, but not Stat3^KO mice following local application of DNFB. Ear thickness was measured at 24, 48 and 72 h following DNFB treatment in mice that had been sensitized by DNFB pre-treated on the abdomen 5 days earlier (mean ± SEM; WT, N = 15; Stat5ab^KO/+, N = 13; Stat3^KO/+, N = 10; Stat5ab^KO/Stat3^KO, N = 6; *p < 0.05, **p < 0.01, ***p < 0.001).

2. B–D. Significant reduction in peripheral blood (C) CD8+, but not (D) CD4+ T cells or (B) total white blood cells (mean ± SEM; WT, N = 9; Stat5ab^KO/+, N = 9; Stat3^KO/+, N = 7; Stat5ab^KO/Stat3^KO, N = 6; *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 10. Contact hypersensitivity and metabolic parameters in Dp11(1)/+; Stat5ab ^KO /+ double mutant mice

1. A,B. The significant (A) increase in total white blood cells and (B) decrease in baseline glucose seen in Dp11(1)/+ mice compared to WT mice was reversed in Dp11(1)/+;Stat5ab^KO/+ double mutant littermates (Dp11(1), N = 7; Dp11(1)/Stat5ab, N = 9; WT, N = 10; Stat5ab, N = 8).

2. C. The trend for increased glucose tolerance seen in Dp11(1)/+ mice compared to WT mice was reversed in Dp11(1)/+;Stat5ab^KO/+ double mutant littermates (Dp11(1), N = 7; Dp11(1)/Stat5ab, N = 9; WT, N = 14; Stat5ab, N = 7; mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001).