Inter-individual differences in habitual sleep timing and entrained phase of endogenous circadian rhythms of BMAL1, PER2 and PER3 mRNA in human leukocytes

Abstract

Study objectives: Individual sleep timing differs and is governed partly by circadian oscillators, which may be assessed by hormonal markers, or by clock gene expression. Clock gene expression oscillates in peripheral tissues, including leukocytes. The study objective was to determine whether the endogenous phase of these rhythms, assessed in the absence of the sleep-wake and light-dark cycle, correlates with habitual sleep-wake timing.

Design: Observational, cross-sectional.

Setting: Home environment and Clinical Research Center.

Participants: 24 healthy subjects aged 25.0 +/- 3.5 (SD) years.

Measurements: Actigraphy and sleep diaries were used to characterize sleep timing. Circadian rhythm phase and amplitude of plasma melatonin, cortisol, and BMAL1, PER2, and PER3 expression were assessed during a constant routine.

Results: Circadian oscillations were more robust for PER3 than for BMAL1 or PER2. Average peak timings were 6:05 for PER3, 8:06 for PER2, 15:06 for BMAL1, 4:20 for melatonin, and 10:49 for cortisol. Individual sleep-wake timing correlated with the phases of melatonin and cortisol. Individual PER3 rhythms correlated significantly with sleep-wake timing and the timing of melatonin and cortisol, but those of PER2 and BMAL1 did not reach significance. The correlation between sleep timing and PER3 expression was stronger in individuals homozygous for the variant of the PER3 polymorphism that is associated with morningness.

Conclusions: Individual phase differences in PER3 expression during a constant routine correlate with sleep timing during entrainment. PER3 expression in leukocytes represents a useful molecular marker of the circadian processes governing sleep-wake timing.

Figure 1

Habitual sleep timing. Mean sleep onset, mean sleep mid-point (± SD), and mean wake times are shown for each individual (gray bars), ordered by sleep mid-point. • = PER3^4/4 subjects, ▴ = PER3^5/5 subjects.

Figure 2

Individual and average oscillations in circadian markers. Left Panel: z-score normalized rhythms from individual subjects for PER2, BMAL1, PER3, melatonin and cortisol plotted relative to clock time (data for clock genes are the relative copy number/GAPDH ratio). Right Panel: Average z-score curves for each marker for all individuals (± SD). Mean sleep onset and wake times are depicted as a bar above the melatonin profile (± SD).

Figure 3

Characterization of circadian marker rhythms. (A) Percentage of individuals in whom, under constant routine conditions, a significant amplitude could be detected for each circadian marker. The actual number of individuals is shown above each bar. (B) Mean fitted z-score amplitudes (± SD). (C) Mean fitted z-score phase maxima (± SD). **P = 0.0027, ***P = 0.0004. CORT = Cortisol, MEL = Melatonin

Figure 4

Scatter plots showing the correlations between PER3 expression and sleep markers for each individual (represented by each dot). The mean fitted z-score peak (time in hours) of PER3 expression is plotted against (A) sleep onset (r = 0.48, P = 0.03), (B) wake time (r = 0.44, P = 0.055), (C) mid-sleep time (r = 0.49, P = 0.03), and the mean fitted z-score melatonin onset (D) (r = 0.59, P = 0.005) (all times in hours). Also shown are the correlations for melatonin onset against sleep time (E) (r = 0.78, P < 0.0001) and wake time (F) (r = 0.80, P < 0.0001).

Supplementary Figure S1

Individual clock gene oscillations. Relative copy number/GAPDH ratio data for PER3, PER2, and BMAL1 for each individual plotted against time in hours. The data have been fitted as described in the methods section.