Two novel mouse models mimicking minor deletions in 22q11.2 deletion syndrome revealed the contribution of each deleted region to psychiatric disorders

Abstract

22q11.2 deletion syndrome (22q11.2DS) is a disorder caused by the segmental deletion of human chromosome 22. This chromosomal deletion is known as high genetic risk factors for various psychiatric disorders. The different deletion types are identified in 22q11.2DS patients, including the most common 3.0-Mb deletion, and the less-frequent 1.5-Mb and 1.4-Mb deletions. In previous animal studies of psychiatric disorders associated with 22q11.2DS mainly focused on the 1.5-Mb deletion and model mice mimicking the human 1.5-Mb deletion have been established with diverse genetic backgrounds, which resulted in the contradictory phenotypes. On the other hand, the contribution of the genes in 1.4-Mb region to psychiatric disorders is poorly understood. In this study, we generated two mouse lines that reproduced the 1.4-Mb and 1.5-Mb deletions of 22q11.2DS [Del(1.4 Mb)/+ and Del(1.5 Mb)/+] on the pure C57BL/6N genetic background. These mutant mice were analyzed comprehensively by behavioral tests, such as measurement of locomotor activity, sociability, prepulse inhibition and fear-conditioning memory. Del(1.4 Mb)/+ mice displayed decreased locomotor activity, but no abnormalities were observed in all other behavioral tests. Del(1.5 Mb)/+ mice showed reduction of prepulse inhibition and impairment of contextual- and cued-dependent fear memory, which is consistent with previous reports. Furthermore, apparently intact social recognition in Del(1.4 Mb)/+ and Del(1.5 Mb)/+ mice suggests that the impaired social recognition observed in Del(3.0 Mb)/+ mice mimicking the human 3.0-Mb deletion requires mutations both in 1.4-Mb and 1.5 Mb regions. Our previous study has shown that Del(3.0 Mb)/+ mice presented disturbance of behavioral circadian rhythm. Therefore, we further evaluated sleep/wakefulness cycles in Del(3.0 Mb)/+ mice by electroencephalogram (EEG) and electromyogram (EMG) recording. EEG/EMG analysis revealed the disturbed wakefulness and non-rapid eye moving sleep (NREMS) cycles in Del(3.0 Mb)/+ mice, suggesting that Del(3.0 Mb)/+ mice may be unable to maintain their wakefulness. Together, our mouse models deepen our understanding of genetic contributions to schizophrenic phenotypes related to 22q11.2DS.

Keywords: 22q11.2 deletion syndrome; Behavioral analysis; Mouse models; Rare deletion types; Sleep analysis.

Fig. 1

Generation of Del(1.4 Mb)/+ and Del(1.5 Mb)/+ mice. a Schematic diagram showing the mouse chromosome 16qA13 that is syntenic region of human chromosome 22q11.2. Green boxes represent low copy repeats (LCRs). Each black square represents one gene which is conserved between human and mouse. Pink squares represent non-conserved genes. Blue, red, and green horizontal bars indicate the human hemizygous genomic deletions (3.0-Mb, 1.5-Mb and 1.4-Mb, respectively) and syntenic mouse genomic regions [Del(3.0 Mb)/+, Del(1.5 Mb)/+ and Del(1.4 Mb)/+]. b Schematic illustration of wild-type (WT) and Del(1.4 Mb)/+ alleles. Green arrows, sgRNA sites; pink bars, bridging single-stranded oligodeoxyribonucleotides (ssODNs); gray squares, Pi4ka exons; white squares, Dgcr2 exons; black arrowheads, PCR primers; dashed line, deleted region in the Del(1.4 Mb)/+ allele. c Schematic illustration of WT and Del(1.5 Mb)/+ alleles. Green arrows, sgRNA sites; pink bars, bridging ssODNs; white squares, Dgcr2 exons; black squares, Hira exons; black arrowheads, PCR primers; dashed line, deleted region in the Del(1.5 Mb)/+ allele. d A representative result of genotyping PCR analysis of Del(1.4 Mb)/+ founder candidates. P.C., positive control. e Genotyping analysis of N1 offspring of Del(1.4 Mb)/+ founder × WT crossing. f A representative result of genotyping PCR analysis of Del(1.5 Mb)/+ founder candidates. P.C., positive control. g Genotyping analysis of N1 offspring of Del(1.5 Mb)/+ founder × WT crossing. h The nucleotide sequencing analysis of PCR-amplified fragments around the deletion junction in Del(1.4 Mb)/+ founder candidates. sgRNA sites are indicated in bold and protospacer adjacent motif (PAM) sites in red. A mismatch nucleotide is indicated in blue. The expected sgRNA-guided cutting sites are indicated by pink arrows. i Deletion junction sequences of Del(1.5 Mb)/+ founder candidates. sgRNA sites are indicated in bold and PAM sites in red. The expected sgRNA-guided cutting sites are indicated by pink arrows

Fig. 2

Detection of DNA copy number changes and quantification of mRNA expression in the brain. a, b Array CGH profiles of chromosome 16qA13 from N1 Del(1.4 Mb)/+ (a) and Del(1.5 Mb)/+ mice (b) showing the decrease in copy number of targeting regions (pink). Array CGH data are displayed in a magnified view. c Quantitative RT-PCR analysis of mRNA from the hippocampi of WT (n = 5) and Del(1.4 Mb)/+ (n = 5) mice. d Quantitative RT-PCR analysis of WT (n = 5) and Del(1.5 Mb)/+ (n = 5) mRNA. Data are expressed as means ± SEM

Fig. 3

General locomotor activity in the open field test. a, b Performance in open field test of Del(1.4 Mb)/+ mice (n = 12 for WT; n = 12 for Del(1.4 Mb)/+ mice). a Total distance moved during the 10 min test period. b Percentage of time spent in the outer zone. c, d Performance in open field test of Del(1.5 Mb)/+ mice (n = 11 for WT; n = 12 for Del(1.5 Mb)/+ mice). c Total distance moved during the 10 min test period. d Percentage of time spent in the outer zone. Data are expressed as mean ± SEM. n.s. not significant; **p < 0.01

Fig. 4

Performance in five-trial direct social interaction test. a Performance in five-trial direct social interaction test of Del(1.4 Mb)/+ mice (n = 12 for WT; n = 12 for Del(1.4 Mb)/+ mice). b Performance in five-trial direct social interaction test of Del(1.5 Mb)/+ mice (n = 11 for WT; n = 12 for Del(1.5 Mb)/+ mice). Data are expressed as mean ± SEM. n.s. not significant

Fig. 5

Prepulse inhibition (PPI) analysis of the acoustic startle response. a Percentage of PPI in Del(1.4 Mb)/+ mice (n = 12 for WT; n = 12 for Del(1.4 Mb)/+ mice). b Measurement of acoustic startle response to the 120-dB startle stimulus in Del(1.4 Mb)/+ mice. c Percentage of PPI in Del(1.5 Mb)/+ mice (n = 11 for WT; n = 12 for Del(1.5 Mb)/+ mice). d Measurement of acoustic startle response to the 120-dB startle stimulus in Del(1.5 Mb)/+ mice. n.s. not significant; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Contextual and cued fear-conditioning test. a Fear-conditioning test of Del(1.4 Mb)/+ mice (n = 12 for WT; n = 12 for Del(1.4 Mb)/+ mice). Context-dependent freezing response (%) towards the tone and foot-shock paring (conditioning) measured 24 h after the initial exposure (left panel). Cued-dependent freezing response (%) measured 48 h after the conditioning (right panel). b Fear-conditioning test of Del(1.5 Mb)/+ mice (n = 11 for WT; n = 12 for Del(1.5 Mb)/+ mice). Context-dependent freezing response (%) (left panel). Cued-dependent freezing response (%) (right panel). Data are expressed as mean ± SEM. n.s. not significant; ***p < 0.001

Fig. 7

Sleep and wakefulness cycle in Del(3.0 Mb)/+ mice. a–c Circadian variation in wakefulness (a), NREM sleep (b) and REM sleep (c) in Del(3.0 Mb)/+ mice (n = 9 for WT; n = 16 for Del(3.0 Mb)/+ mice). Data are expressed as minutes per hour spent in each stage, averaged from EEG/EMG recordings during two consecutive 24-h periods. d Total time of wakefulness, NREM sleep and REM sleep stages in 24-h period, light period (L) and dark period (D). e Episode duration of wakefulness, NREM sleep and REM sleep stages in 24-h period, light period and dark period. f REM latency in 24-h period, light period and dark period. g REM interval in 24-h period, light period and dark period. h Values indicate the number of transitions between wakefulness, NREM sleep and REM sleep per 24 h. i–k EEG spectral profiles of WT (blue line, n = 9) and Del(3.0 Mb)/+ mice (red line, n = 16) during wakefulness (i), NREM sleep (j) and REM sleep (k). The average EEG spectra were normalized to total EEG power from 1 to 32 Hz in 1 Hz bins. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001