Deletion of the Snord116/SNORD116 Alters Sleep in Mice and Patients with Prader-Willi Syndrome

Abstract

Study objectives: Sleep-wake disturbances are often reported in Prader-Willi syndrome (PWS), a rare neurodevelopmental syndrome that is associated with paternally-expressed genomic imprinting defects within the human chromosome region 15q11-13. One of the candidate genes, prevalently expressed in the brain, is the small nucleolar ribonucleic acid-116 (SNORD116). Here we conducted a translational study into the sleep abnormalities of PWS, testing the hypothesis that SNORD116 is responsible for sleep defects that characterize the syndrome.

Methods: We studied sleep in mutant mice that carry a deletion of Snord116 at the orthologous locus (mouse chromosome 7) of the human PWS critical region (PWScr). In particular, we assessed EEG and temperature profiles, across 24-h, in PWScr (m+/p-) heterozygous mutants compared to wild-type littermates. High-resolution magnetic resonance imaging (MRI) was performed to explore morphoanatomical differences according to the genotype. Moreover, we complemented the mouse work by presenting two patients with a diagnosis of PWS and characterized by atypical small deletions of SNORD116. We compared the individual EEG parameters of patients with healthy subjects and with a cohort of obese subjects.

Results: By studying the mouse mutant line PWScr(m+/p-), we observed specific rapid eye movement (REM) sleep alterations including abnormal electroencephalograph (EEG) theta waves. Remarkably, we observed identical sleep/EEG defects in the two PWS cases. We report brain morphological abnormalities that are associated with the EEG alterations. In particular, mouse mutants have a bilateral reduction of the gray matter volume in the ventral hippocampus and in the septum areas, which are pivotal structures for maintaining theta rhythms throughout the brain. In PWScr(m+/p-) mice we also observed increased body temperature that is coherent with REM sleep alterations in mice and human patients.

Conclusions: Our study indicates that paternally expressed Snord116 is involved in the 24-h regulation of sleep physiological measures, suggesting that it is a candidate gene for the sleep disturbances that most individuals with PWS experience.

Keywords: Prader-Willi; Snord116; sleep; temperature; theta rhythms.

© 2016 Associated Professional Sleep Societies, LLC.

Figures

Figure 1

Human SNRPN cluster and mouse orthologous Snrpn cluster (not to scale). (A) Representation of the deletion of Patient 1 and Patient 2. Patient 1 carries an atypical deletion with the proximal breakpoint at RP11-484P15 and the distal breakpoint located in the common BP4. The size of the deletion is 6, 27 Mb. Patient 2 exhibits a deletion with the proximal breakpoint at base 22,648,348 and the distal one at base 23,020,695 of chromosome 15. The size of the deletion is 372 Kb. (B) Human SNRPN cluster of chromosome 15 and (C) the orthologues Snrpn cluster of chromosome 7C in mice. Pink rectangles are maternally expressed genes, blue rectangles are paternally expressed genes; when both pink and blue rectangles are present, genes are expressed biallelically. Gene expression/imprinting profiles are reported only for the brain. C, centromere; T, telomere; note that genes are in the same order in human and mice chromosomes but in reverse orientation. Dashed line links homologues. (D) Representation of the deletion of the Prader-Willi syndrome critical region including Snord116 and IPW exons A1/A2, B, and C carried by the murine model PWScrm+/p− studied in this investigation.

Figure 2

Sleep profiling in PWScrm+/p− mutant mice versus controls. (A) Example of electroencephalographic traces (i.e. electroencephalography and electromyography) scored as wakefulness, nonrapid eye movement (NREM) sleep, rapid eye movement (REM) sleep, and REM intrusions. Fast Fourier transforms (FFTs) are represented for each epoch in which delta (light gray bars) and theta (black bars) are plotted. (B) Total time spent asleep per hour in NREM and REM sleep across the 24 h in PWScrm+/p− mice versus controls. (C) Amount of episodes of sleep, NREM and REM sleep, per hour, across the 24 h in PWScrm+/p− mice versus controls. An episode is intended as one or more consecutive epochs of the same stage (i.e. wake [W], NREM sleep or REM sleep). (D) REM sleep intrusions during wakefulness in mutants versus control mice. (E) Power densities of the whole spectrum of frequencies and detailed histograms for delta and theta EEG frequencies (μV) in NREM and REM sleep in mutants versus control mice in the light phase. (F) Peripheral temperature of PWScrm+/p− mice and controls across 24 h. Histograms show the sleep and temperature means ± standard error of the mean (SEM), in the light and dark phases, of the two genotypes. All graphs are presented as mean ± SEM across 24 h. Light and dark phases are indicated by the white and black strips on top of each graph. ZT, Zeitgeber time. Statistical significance is represented as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Sleep in patients with Prader-Willi syndrome compared to healthy and obese control groups. Boxplots of percentages of nonrapid eye movement (NREM) and rapid eye movement (REM) sleep, number of REM sleep cycles and REM sleep episodes for healthy controls (A) and obese subjects (B); colored circles indicate the outlier position of the two patients respect to the control distributions. (C) Power densities of the whole spectrum of frequencies and detailed histograms for delta and theta electroencephalographic frequencies (μV) in NREM and REM sleep in patients, healthy and obese individuals. Graphs are presented as mean ± standard error of the mean; **P < 0.01.

Figure 4

Magnetic resonance imaging analyses in PWScrm+/p− mice compared to wild-type littermate controls. Representative coronal (A–C), horizontal (D), and sagittal (E) slice reconstruction of the areas showing a statistically significant decrease in gray-matter volume (GMV) in PWScrm+/p− mice compared with control littermates. Decrease of gray-matter volume was evident in the vHPC, DG (A,B), vDB (C) and MS (D,E). The statistical significance is indicated by blue color coding (P < 0.05).The arrows indicate the placement of region of interest for post hoc analyses in the vHPC, MS, and vDB areas. Bar graphs (F) illustrate the mean and standard deviation of GMV of dHPC, vHPC, MS, and vDB plotted as a function of genotype. vHPC, ventral hippocampus; dHPC, dorsal hippocampus; MS, medial septal nuclei; vDB, ventral nuclei of the diagonal band; GMV, gray matter volume. Statistical significance is represented as *P < 0.05.