Phenotypic consequences of copy number variation: insights from Smith-Magenis and Potocki-Lupski syndrome mouse models

Abstract

A large fraction of genome variation between individuals is comprised of submicroscopic copy number variation of genomic DNA segments. We assessed the relative contribution of structural changes and gene dosage alterations on phenotypic outcomes with mouse models of Smith-Magenis and Potocki-Lupski syndromes. We phenotyped mice with 1n (Deletion/+), 2n (+/+), 3n (Duplication/+), and balanced 2n compound heterozygous (Deletion/Duplication) copies of the same region. Parallel to the observations made in humans, such variation in gene copy number was sufficient to generate phenotypic consequences: in a number of cases diametrically opposing phenotypes were associated with gain versus loss of gene content. Surprisingly, some neurobehavioral traits were not rescued by restoration of the normal gene copy number. Transcriptome profiling showed that a highly significant propensity of transcriptional changes map to the engineered interval in the five assessed tissues. A statistically significant overrepresentation of the genes mapping to the entire length of the engineered chromosome was also found in the top-ranked differentially expressed genes in the mice containing rearranged chromosomes, regardless of the nature of the rearrangement, an observation robust across different cell lineages of the central nervous system. Our data indicate that a structural change at a given position of the human genome may affect not only locus and adjacent gene expression but also "genome regulation." Furthermore, structural change can cause the same perturbation in particular pathways regardless of gene dosage. Thus, the presence of a genomic structural change, as well as gene dosage imbalance, contributes to the ultimate phenotype.

Figure 1. SMS and PTLS mouse models.

Schematic representation of the mouse chromosome 11 B2 region syntenic to the SMS and PTLS critical region to compare the genotypes of the four strains used in this report (adapted from [34]). Only a few genes of the engineered region are displayed. The region contains the following loci, whose expression is profiled by 70 different probesets: Cops3, Nt5m, Med9, Rasd1, Pemt, Rai1, Srebf1, Tom1l2, Lrrc48, Atpaf2, 4933439F18Rik, Drg2, Myo15, Alkbh5, AW215868, Llgl1, Flii, Smcr7, Top3a, Smcr8, Shmt1, Dhrs7b, Tmem11, Gtlf3b, Gtlf3a, Map2k3, Kcnj12, Tnfrsf13b, Usp22, Aldh3a1, Aldh3a2, Slc47a2, Slc47a1, and Zfp179 (a.k.a. Rnf112) (for GeneIDs, see Materials and Methods). The Cops3 and Zfp179 loci were used as anchoring points to engineer the rearrangement , thus their number of copies does not correlate with the number of copies of the region. Furthermore, some copies of Cops3 (indicated by an X) were inactivated in the process .

Figure 2. Anxiety in the plus maze test is not normalized with the correction of the gene copy number.

The percentage of observations in each arm or the center of the plus maze is represented. Light grey columns: Dp(11)17/+ (N = 19); dark grey columns: Df(11)17/+ (N = 20); white columns: +/+ (N = 27); and black columns: Df(11)17/Dp(11)17 (N = 22). Values represent mean ± SEM. The asterisk denotes significant differences (* p<0.05).

Figure 3. Some social behaviors are dependent on the presence of genomic rearrangements.

(A) Percentage of observations in the chamber side with stranger 1 (Stg1, white columns) or with the empty container (EC, black columns) during the sociability test is shown for the four different groups of mice. (B) Percentage of observations in the chamber side with stranger 1 (Stg1, white columns) or with stranger 2 (Stg2, grey columns) during the preference for social novelty test is depicted. For each genotype the number of mice tested was: N = 21 for Dp(11)17/+, N = 23 for Df(11)17/+, N = 28 for +/+, and N = 22 for Df(11)17/Dp(11)17 mice. The mean ± S.E.M. values are presented. Asterisk denotes significantly differences (* p<0.05).

Figure 4. Differentially expressed genes in SMS and PTLS mouse models.

Distribution of the mapping regions of the top 100 (A) and top 1,000 ranked (B) most differentially expressed transcripts in the cerebellum (C), heart (H), kidney (K), testis (T), and hippocampus (Hi) or present on the array (Affy) of Df(11)17/+ (SMS model, 1n), Dp(11)17/+ (PTLS model, 3n), Df(11)17/Dp(11)17 (2n compound heterozygote), and +/+ (2n) mice (Most-diff dataset, see Figure 1 for a schematic representation of the mouse 11 B2 region of the different mouse models). Proportion of transcripts mapping to the SMS/PTLS rearranged interval (purple), the remainder of mouse chromosome 11 (burgundy), and elsewhere (yellow). Transcripts mapping to the rearranged interval and to the remainder of mouse chromosome 11 are both statistically overrepresented in all tested tissues (all p<1×10⁻⁴). Heatmap of the changes in expression levels of the 49 Most-diff transcripts mapping to the SMS/PTLS rearranged interval (C) and the remainder of mouse chromosome 11 (81 transcripts) (D) measured in Df(11)17/+ (d), Dp(11)17/+ (D), and Df(11)17/Dp(11)17 (dD) mice as compared to +/+ individuals in cerebellum (C), heart (H), kidney (K), testis (T), and hippocampus (Hi). The arrowhead and asterisk denote Cops3 and Zfp179 transcripts, respectively. These transcripts were used as anchors in the strain engineering process, thus they are not present in the same number of copies than other SMS/PTLS genes in the mice models (see Figure 1 and text for details).