Transcript level alterations reflect gene dosage effects across multiple tissues in a mouse model of down syndrome

Abstract

Human trisomy 21, which results in Down syndrome (DS), is one of the most complicated congenital genetic anomalies compatible with life, yet little is known about the molecular basis of DS. It is generally accepted that chromosome 21 (Chr21) transcripts are overexpressed by about 50% in cells with an extra copy of this chromosome. However, this assumption is difficult to test in humans due to limited access to tissues, and direct support for this idea is available for only a few Chr21 genes or in a limited number of tissues. The Ts65Dn mouse is widely used as a model for studies of DS because it is at dosage imbalance for the orthologs of about half of the 284 Chr21 genes. Ts65Dn mice have several features that directly parallel developmental anomalies of DS. Here we compared the expression of 136 mouse orthologs of Chr21 genes in nine tissues of the trisomic and euploid mice. Nearly all of the 77 genes which are at dosage imbalance in Ts65Dn showed increased transcript levels in the tested tissues, providing direct support for a simple model of increased transcription proportional to the gene copy number. However, several genes escaped this rule, suggesting that they may be controlled by additional tissue-specific regulatory mechanisms revealed in the trisomic situation.

Figure 1

Boundaries of the proximal T(16;17)65Dn breakpoint were determined by FISH. Left: Schematic illustrating the synteny between HSA21 and MMU16, and the translocation chromosome MMU17¹⁶ with the genes at the boundaries of the triplicated segment. Chromosomal position of the two BACs used for the FISH analysis is indicated. Right: Metaphase spreads from T(16;17)65Dn mouse; BAC 134F19 (top) is in red and BAC 359P19 (bottom) is in green. Arrows point to the MMU17¹⁶ chromosome.

Figure 2

Functional distribution of the 136 mmu21 genes in nine tissues of euploid mice. The 136 Chr21 orthologs (mmu21) were distributed into 10 categories of biological processes (left bar; Hill et al. 2002). Bars show the functional distribution of the genes expressed in each tissue, as percent of the total number of mmu21 genes. The second left bar (all brain) represents the accumulated data from the three brain regions.

Figure 3

Hierarchical clustering showing the expression levels of mmu21 genes across nine tissues of the control mice and Ts65Dn mice. For each clone we calculated the logarithm (base 2) of the ratio between the normalized intensity in the specific tissue and the average of intensities of this clone across the nine control tissues. Spot intensities below the average of intensities across all tissues give log-ratio values ranging from –12.6 to 0, and are represented by a color gradient spanning from light to dark green. Conversely, spot intensities above the average of intensities across all tissues give log-ratio values ranging from 0 to +12.6, and are represented by a color gradient spanning from dark to light red. Thirty-one clones (24 genes) that did not show significant expression values across all control tissues were excluded from the clustering; 130 clones (112 genes; rows) and nine tissues of control and TS65Dn mice respectively (columns) were clustered using the average-linkage hierarchical clustering method with Pearson correlation as similarity measure (J-Express V 2.1; www.molmine.com). Additionally, clones with the most similar expression profiles to Dscam (with respect to the Pearson correlation) are displayed: 10% closest (13 clones, left column), 15% closest (20 clones, middle column), and 20% closest (26 clones, right column). Note that in hierarchical clustering procedures, clones with similar expression profiles can be split to different parts of the dendrogram (e.g., Olig2) and vice-versa (e.g., Abcg1). Genes referenced in the text are highlighted as follows: *, Sh3bgr; Black bars: A: Group of genes highly expressed in the kidney and the liver (from Ifnar 2 to Pttg1ip, P-value = 1.05 10^–4) of controls and trisomics. B: Group of genes with predominant expression in the testis of controls and trisomics. C: Cluster of genes highly expressed in the kidney and the liver (from Col18a1 to Ftcd, P-value = 5.84 10^–3) of controls and trisomics. An interactive version of Figure 3 is given in Supplemental Figure 3.

Figure 4

Linear regression plots comparing trisomic and control samples. For each plot corresponding to a given tissue, the linear regression for the triplicated genes is in green, and that of the UniGene sample is in blue. Each clone was plotted using the average of its normalized spot intensities obtained in the replicated hybridizations, with the value in control tissue on the x-axis and with the value in Ts65Dn tissue on the y-axis. Bars show the interval [μ–σ,μ+σ] where μ and σ are the mean and standard deviation, respectively, for each data point across the replicated experiments. We excluded from the graphics outlier spots with intensities ±4 std. dev. above the mean intensity (e.g., Wrb). (S) is the slope for the regression line, (I) the intercept, and (C) the correlation. Scales range from 0–100.000, except for lung and skeletal muscle, which range from 0–50.000.

Figure 5

Comparison of trisomic/euploid ratios obtained by real-time PCR and arrays for the three brain regions. Histograms showing the correlation between array and qPCR results for cerebellum, cortex, and midbrain. Blue bars represent mean ratios of three independent array hybridizations, and red bars represent mean ratios of two independent qPCR experiments. Error bars were calculated by averaging standard deviations of the independent experiments. Genes represented by 1–3 clones on the arrays were independently compared to the qPCR result. (*) beside a gene name indicates an array ratio (blue bar) with a significant P-value (<0.05). Correlation histograms for all tissues are available in Supplemental Figure 2.