Generation of the Sotos syndrome deletion in mice

Abstract

Haploinsufficiency of the human 5q35 region spanning the NSD1 gene results in a rare genomic disorder known as Sotos syndrome (Sotos), with patients displaying a variety of clinical features, including pre- and postnatal overgrowth, intellectual disability, and urinary/renal abnormalities. We used chromosome engineering to generate a segmental monosomy, i.e., mice carrying a heterozygous 1.5-Mb deletion of 36 genes on mouse chromosome 13 (4732471D19Rik-B4galt7), syntenic with 5q35.2-q35.3 in humans (Df(13)Ms2Dja ( +/- ) mice). Surprisingly Df(13)Ms2Dja ( +/- ) mice were significantly smaller for their gestational age and also showed decreased postnatal growth, in contrast to Sotos patients. Df(13)Ms2Dja ( +/- ) mice did, however, display deficits in long-term memory retention and dilation of the pelvicalyceal system, which in part may model the learning difficulties and renal abnormalities observed in Sotos patients. Thus, haploinsufficiency of genes within the mouse 4732471D19Rik-B4galt7 deletion interval play important roles in growth, memory retention, and the development of the renal pelvicalyceal system.

Fig. 1

Generation of a 1.5-Mb deletion between the 4732471D19Rik and B4galt7 loci. a Schematic representation of HSA5 and the syntenic region on MMU13 with the end points of the syntenic regions indicated (4732471D19Rik and B4galt7). Genes that map to the human 5q35.2–q35.3 region (NCBI build GRChr37) and the B1 band on MMU13 (NCBI build m37) are listed in Supplementary Table 1. b The targeting vectors used to generate the 1.5-Mb deletion between the 4732471D19Rik and B4galt7 loci contained a loxP site (arrowhead), a selectable antibiotic resistance gene [N (neomycin) or P (puromycin)], a coat-colour marker (Ty or Ag), and part of the Hprt gene (5′ or 3′), which were targeted as indicated (B, BstEII; S, SpeI; P, puromycin; N, neomycin; Ty, Tyrosinase; Ag, Agouti). c Comparative genomic hybridization of the Sotos deletion interval. d Interphase FISH analysis with BAC probes that map either inside (green) or outside of (red) the deletion interval. Chromosomes from the ES cells double-targeted in cis (Df(13)Ms2Dja ^+/−) showed a red signal indicating deletion of the 4732471D19Rik-B4galt7 interval (*), while the wild-type chromosome showed red and green signals (**). There are two additional green centromeric signals present due to nonspecific binding of the probe

Fig. 2

Phenotypic analysis of Df(13)Ms2Dja ^+/− mice at various time points. a Body weight measurements in control (+/+, n = 12) and deficiency (Df(13)Ms2Dja ^+/−, n = 12) littermates at embryonic day 15.5. b Body weight measurements in control (+/+, n = 25) and deficiency (Df(13)Ms2Dja ^+/−, n = 21) littermates at 1 day of age. c, d Body weight and length measurements in control (+/+, n = 12) and deficiency (Df(13)Ms2Dja ^+/−, n = 11) littermates at 28 weeks of age. e, f DEXA analysis of lean mass and fat mass in control (+/+, n = 12) and deficiency (Df(13)Ms2Dja ^+/−, n = 11) littermates at 28 weeks of age. All data were statistically analysed using the two-tailed Student’s t-test, and the asterisk indicates statistical significance. The error bars represent the standard deviation of the measurements

Fig. 3

Learning impairment in Df(13)Ms2Dja ^+/− mice. a Habituation-dishabituation test. Both deficiency (Df(13)Ms2Dja ^+/−, n = 9) and control (+/+, n = 8) mice recognized two different stimulus animals, as shown by a decline in the investigation time over trials 1–4 when they were repetitively presented the same stimulus animal (mouse A), and an increase in the investigation time on trial 5 when they were presented with a novel stimulus animal (mouse B) (trial 4 vs. trial 5, P < 0.0001, post-hoc analysis after two-way ANOVA). b Discrimination test (performed 24 h after the habituation-dishabituation test). Df(13)Ms2Dja ^+/− mice spent significantly more time investigating the familiar stimulus mouse (mouse A) than did control mice (P < 0.05, two-tailed Student’s t-test), suggesting that Df(13)Ms2Dja ^+/− mice were less able to distinguish a familiar from an unfamiliar animal (mouse C). The error bars represent the standard error of the mean and the asterisks indicate statistical significance. Four animals (one deficiency and three wild types) were withdrawn from the experiment because of their low investigation times (less than 10 s on trial 1)

Fig. 4

Dilation of the renal pelvicalyceal system in Df(13)Ms2Dja ^+/− mice. Haematoxylin and eosin-stained kidney sections from control (+/+) and deficiency (Df(13)Ms2Dja ^+/−) mice at a 10 weeks, b 28 weeks, and c 52 weeks of age. Kidneys of deficiency (Df(13)Ms2Dja ^+/−) mice showed mild through moderate to severe dilation of the pelvicalyceal system (left to right) (visible as empty spaces; indicated by asterisks) compared with control littermates (normal structure of the pelvicalyceal system indicated by asterisks). Images are representative and taken at ×250 magnification