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. Observational, cross-sectional. Home environment and Clinical Research Center. 24 healthy subjects aged 25.0 ± 3.5 (SD) years. 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. 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 signifcance. 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. 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.

To screen the PER3 promoter for polymorphisms and investigate the phenotypic associations of these polymorphisms with diurnal preference, delayed sleep phase disorder/syndrome (DSPD/DSPS), and their effects on reporter gene expression. Interspecific comparison was used to define the approximate extent of the PER3 promoter as the region between the transcriptional start site and nucleotide position -874. This region was screened in DNA pools using PCR and direct sequencing, which was also used to screen DNA from individual participants. The different promoter alleles were cloned into a luciferase expression vector and a deletion library created. Promoter activation was measured by chemiluminescence. N/A DNA samples were obtained from volunteers with defined diurnal preference (3 x 80, selected from a pool of 1,590), and DSPD patients (n = 23). N/A We verified three single nucleotide polymorphisms (G −320T, C −319A, G −294A), and found a novel variable number tandem repeat (VNTR) polymorphism (-318 1/2 VNTR). The -320T and -319A alleles occurred more frequently in DSPD compared to morning (P = 0.042 for each) or evening types (P = 0.006 and 0.033). The allele combination TA2G was more prevalent in DSPD compared to morning (P = 0.033) or evening types (P = 0.002). Luciferase expression driven by the TA2G combination was greater than for the more common GC2A (P < 0.05) and the rarer TA1G (P < 0.001) combinations. Deletion reporter constructs identified two enhancer regions (−703 to −605, and −283 to −80). Polymorphisms in the PER3 promoter could affect its expression, leading to potential differences in the observed functions of PER3.

Summary: The objective of this study was to investigate the effect of age on the association between preferred timing of sleep and waking activities and a coding‐region variable number tandem repeat (VNTR) polymorphism in the clock gene PER3. We have previously reported this polymorphism to associate with diurnal preference and delayed sleep phase syndrome (DSPS). Participants (n = 1590; 707 males and 883 females) completed the Horne–Östberg (HÖ) questionnaire for diurnal preference and provided a DNA sample. Overall HÖ scores were plotted against age. The 5% extremes and intermediates were selected for genotyping. Frequencies of the PER3 4‐ and 5‐repeat alleles were examined in separate age groups (18–29, 30–39, 40–49 and 50+ years of age). The 4‐repeat allele was significantly more frequent in evening types, and the 5‐repeat allele more frequent in morning types (Fisher's exact test, P = 0.016). Analysis in the four age groupings revealed that the strength of this association attenuated with age and was significant only in the youngest group (18–29 years). These results extend our previous finding of an association between the PER3 VNTR and diurnal preference. They also demonstrate that diurnal preference in young people is more closely associated with this polymorphism than it is in other age groups.