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. 2021 Dec 6;31(23):5238-5248.e7.
doi: 10.1016/j.cub.2021.09.049. Epub 2021 Oct 14.

The dihydropyrimidine dehydrogenase gene contributes to heritable differences in sleep in mice

Brendan T Keenan  1 , Raymond J Galante  1 , Jie Lian  1 , Lin Zhang  1 , Xiaofeng Guo  1 , Olivia J Veatch  2 , Elissa J Chesler  3 , W Timothy O'Brien  4 , Karen L Svenson  3 , Gary A Churchill  3 , Allan I Pack  5
Affiliations

The dihydropyrimidine dehydrogenase gene contributes to heritable differences in sleep in mice

Brendan T Keenan et al. Curr Biol. .

Abstract

Many aspects of sleep are heritable, but only a few sleep-regulating genes have been reported. Here, we leverage mouse models to identify and confirm a previously unreported gene affecting sleep duration-dihydropyrimidine dehydrogenase (Dpyd). Using activity patterns to quantify sleep in 325 Diversity Outbred (DO) mice-a population with high genetic and phenotypic heterogeneity-a linkage peak for total sleep in the active lights off period was identified on chromosome 3 (LOD score = 7.14). Mice with the PWK/PhJ ancestral haplotype at this location demonstrated markedly reduced sleep. Among the genes within the linkage region, available RNA sequencing data in an independent sample of DO mice supported a highly significant expression quantitative trait locus for Dpyd, wherein reduced expression was associated with the PWK/PhJ allele. Validation studies were performed using activity monitoring and EEG/EMG recording in Collaborative Cross mouse strains with and without the PWK/PhJ haplotype at this location, as well as EEG and EMG recording of sleep and wake in Dpyd knockout mice and wild-type littermate controls. Mice lacking Dpyd had 78.4 min less sleep during the lights-off period than wild-type mice (p = 0.007; Cohen's d = -0.94). There was no difference in other measured behaviors in knockout mice, including assays evaluating cognitive-, social-, and affective-disorder-related behaviors. Dpyd encodes the rate-limiting enzyme in the metabolic pathway that catabolizes uracil and thymidine to β-alanine, an inhibitory neurotransmitter. Thus, data support β-alanine as a neurotransmitter that promotes sleep in mice.

Keywords: Collaborative Cross; Diversity Outbred; Dpyd; dihydropyrimidine dehydrogenase; genetics; knockout; mice; sleep.

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Conflict of interest statement

Declaration of interests A.I.P. is The John L. Miclot Professor of Medicine at the University of Pennsylvania; funds for this endowment were provided by the Philips Respironics Foundation. G.A.C., E.J.C., and K.L.S. disclose that The Jackson Laboratory produces and sells a wide variety of mice for research, including Collaborative Cross and Diversity Outbred mice. The other authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Allele effects at the LOD peak for total sleep during lights off in DO mice.
Founder-specific additive genetic effects on total sleep during lights off are shown in the linkage region identified on chromosome 3 (top panel). The 95% Bayesian credible interval (118.4-122.6 Mb) is denoted with black dashed lines. Colored lines illustrate the relative change in total sleep duration attributable to each of the founder haplotypes, with lines below zero indicating a reduction in total sleep and lines above zero indicating increased total sleep. A large relative reduction in sleep is observed for the PWK/PhJ haplotype (red) in this region, with the NOD haplotype associated with increased sleep (dark blue). LOD scores across the region are shown in the bottom panel. See Figure S1 for candidate genes under LOD peak.
Figure 2.
Figure 2.. Expression of Dpyd in the hippocampus of DO mice.
Founder-specific additive genetic effects on Dpyd expression from RNA sequencing of the hippocampus in an independent sample of DO mice are shown in the top panel. There is a marked reduction in Dpyd expression associated with the PWK/PhJ haplotype at our linkage region of interest, with a LOD score >20. This PWK/PhJ-specific reduction in Dpyd expression is similar to the association seen for total sleep duration during lights off, leading to the selection of Dpyd as the likely causal gene under the linkage peak. Figure and data are available through the online DO Hippocampus QTL Viewer (https://churchilllab.jax.org/qtlviewer/DO/hippocampus). See Figure S1 for founder effects for expression of all genes within the linkage region.
Figure 3.
Figure 3.. Comparison of total sleep during lights off in Collaborative Cross strains with relevant founder Dpyd alleles.
Figure 3A illustrates total sleep during lights off based on high-throughput phenotyping using beam breaks in Collaborative Cross strains with founder Dpyd alleles. There was a significant difference among strains (p=0.005), including differences between the black (CC003/Unc) and white (CC009/Unc) PWK/PhJ Dpyd strains and both the NOD/ShiLtJ (p=0.006 and 0.008, respectively) and WSB/EiJ (p=0.025 and 0.032, respectively) strains. See Tables S1 and S2 for additional phenotype comparisons. Figure 3B shows total sleep from EEG/EMG recording in 3-hour windows during lights off in the CC strains with PWK/PhJ (CC003/Unc) and NOD/ShiLtJ (CC004/TauUnc) Dpyd alleles. A moderately large difference on average was seen in total sleep over the entire lights off period, but this result did not reach statistical significance (p=0.240; Cohen’s d = −0.71). When examining the lights off period in more detail, significantly less sleep was observed in the PWK/PhJ Dpyd strain during the first 3 hours (7PM-10PM; 41.0±26.4 vs. 80.4±7.7 minutes; p=0.003; Cohen’s d = −2.03). See Tables S3 and S4 for additional phenotype comparisons. Results presented as Mean (95% CI)
Figure 4.
Figure 4.. EEG/EMG total sleep in lights off in male and female Dpyd knockout mice.
Total sleep during the lights off period is shown in Dpyd knockout (KO) mice and wildtype (WT) littermate controls, overall and stratified by sex. Among all mice, there was significantly reduced sleep in the knockout mouse (263.5±92.6 minutes) compared to the wildtype control (341.9±74.0 minutes; p=0.007). The impact of the knockout was similar in male and female mice (interaction p=0.689); statistical significance is impacted by lower sample size for sex-specific analyses. See Tables S6 and S7 for additional phenotype comparisons. Sex x Genotype Interaction p=0.689 ∣ Data Presented as Mean (95% CI)
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